CA2968961A1 - Methods and compositions for 18f-radiolabeling of biologics - Google Patents

Abstract

The invention relates to water soluble 18 F-prosthetic groups and the synthesis and use of 18 F-labeled biological molecules containing the 18 F-prosthetic groups for imaging various processes within the body, for detecting the location of molecules associated with disease pathology, and for monitoring disease progression are disclosed.

Description

REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to United States Provisional Application No.
62/084,366, entitled "Methods and Compositions for 18F-Radiolabeling of Biologics" filed November 25, 2014, the contents of which are hereby incorporated by reference.
FIELD

[0002] The invention relates to 18F-prosthetic groups and the synthesis and use of 18F-labeled compositions for imaging various processes within the body, for detecting the location of molecules associated with disease pathology, and for monitoring disease progression.
BACKGROUND

[0003] Positron emission tomography (PET) is a non-invasive imaging technique that has become one of the most widely used methods in diagnostic medicine and drug development, with high sensitivity (fmoles), high resolution (4-10 mm) and tissue accretion that can be quantitated. The valuable in vivo functional information about biological processes in living subjects provided by PET imaging also provides a unique translational medical advantage in that the same tool can be used both preclinically and clinically.

[0004] PET relies on the design and synthesis of molecules labeled with a positron-emitting radioisotopes including 18F, 64Cu, 11C, 150, 13N, , 66¨a U 68Ga, 76Br, 89Zr, 94MTC, 86Y and 124I. /n vivo, these radiotracers or radioligands emit positrons from the nucleus of the isotope with different energies depending on the isotope used. The energy of the ejected positron controls the average distance that it travels before it collides with an electron resulting in the emission of two 511 keV gamma rays in opposite directions. The gamma rays produced by this positron annihilation event are detected by the PET imaging scanner to produce planar and tomographic images that reveal distribution of the radiotracer as a function of time.
Accordingly, isotopes that are pure positron emitters with low ejection energy isotopes are preferred for PET imaging to minimize the distance traveled by the positron before annihilation and dosimetry problems caused by other emissions such as gamma rays, alpha particles or beta particles.

[0005] In addition, the half-life of the isotope used in PET imaging must be long enough to allow synthesis and analysis of the radiotracer molecule, injection into the patient, in vivo

6 PCT/US2015/062502 localization, clearance from non-target tissues and the production of a clear image. 18F (13+
635 keV 97%, ti/2 110 min) is one of the most widely used PET emitting isotopes because of its low positron emission energy, lack of side emissions and suitable half-life.
[0006] Traditionally, labeling biological molecules, such as peptides and proteins, with 18F
has been challenging due to the harsh conditions (high temperatures, organic solvents and strong basic conditions) required for labeling with this radionuclide. One approach for labeling proteins with 18F is the use of a prosthetic group which can withstand harsh fluorination conditions. Numerous prosthetic groups have been reported (e.g., reviewed in Nucl. Med. Bio. 34:5, 2007), but many of these 18F-labeled prosthetic groups require organic solvents and are not amenable for labeling protein or protein like molecules in aqueous media or have other undesirable properties.

[0007] Accordingly, there is still a continuing need for rapid, simple methods of 18F-labeling targeting moieties, such as proteins and peptides, to produce radiotracer compositions which retain sufficient specific activity and stability for use in in vivo imaging methodologies.
SUMMARY

[0008] The present invention is based is based at least in part on the discovery of an 18F
-labeled prosthetic group that contains a nitro-pyridine linked to a polyethylene glycol (PEG) moiety and a terminal azide, which prosthetic group is water soluble and less volatile than other agents used for 18F-labeling, and that reactions incorporating this 18F-labeled prosthetic group into biological molecules (e.g., peptides and proteins) can be monitored by UV. These advantageous features provide an efficient, rapid and reproducible method of producing 8F-labeled -biomolecules under conditions which retain the biological activity of the biomolecule. In certain embodiments, biomolecules (e.g., peptides and proteins) containing bifunctional conjugating moieties (e.g., with ring constrained alkyne groups, such as bifunctional chelators) form covalent bonds with the terminal azide of the 18F-labeled prosthetic group via a "click" biorthogonal reaction to produce radiolabeled probes that are stable under physiological conditions. The UV absorbance of the resultant product further provides a practical, sensitive and rapid analytical method for determining the radiochemical purity of the product.

[0009] In one aspect, provided herein is a 18F-labeled prosthetic group with the following structure, 18j. F}¨[0 ,, (CH2)2]-143 N
or a pharmaceutically acceptable salt thereof, wherein x is an integer from 1 to 8. In some embodiments, x is an integer from 2 to 6. In some embodiments x is an integer from 3 to 5.
In some embodiments, x is 4. In some embodiments, the [0(CH2)2]x moiety is present in the 1-2, 1-3 or 1-4 configuration relative to the nitrogen on the pyridine ring.
In related embodiments, 18F is attached to the pyridine ortho to the N atom. In some embodiments, the pharmaceutically acceptable salt is selected from the group consisting of fluoro, bromo, chloro and iodo salts. In one embodiment, the salt is a trifluoromethanesulfonate salt. In some embodiments, the 18F-radiolabeled prosthetic group or pharmaceutically acceptable salt is water soluble. In some embodiments, the 18F-radiolabeled prosthetic group or pharmaceutically acceptable salt is nonvolatile.

[00010] In some embodiments, the 18F-radiolabeled compound has the structure ¨[0(CF12)2lx¨N3 N>
or a pharmaceutically acceptable salt thereof, wherein x is an integer from 1 to 8. In some embodiments, x is an integer from 2 to 6. In some embodiments x is an integer from 3 to 5.
In some embodiments, x is 4. In some embodiments, the [0(CH2)2]x moiety is present in the 1-2, 1-3 or 1-4 configuration relative to the nitrogen on the pyridine ring.
In some embodiments, the pharmaceutically acceptable salt is selected from the group consisting of fluoro, bromo, chloro and iodo salts. In one embodiment, the salt is a trifluoromethanesulfonate salt. In some embodiments, the 18F-radiolabeled prosthetic group or pharmaceutically acceptable salt is nonvolatile.

[00011] In some embodiments, the 18F-radiolabeled compound has the structure N
[0(CH2)2]õ¨N3 or a pharmaceutically acceptable salt thereof, wherein x is an integer from 1 to 8. In some embodiments, x is an integer from 2 to 6. In some embodiments x is an integer from 3 to 5.
In some embodiments, x is 4. In some embodiments, the pharmaceutically acceptable salt is selected from the group consisting of fluoro, bromo, chloro and iodo salts. In one embodiment, the salt is a trifluoromethanesulfonate salt. In some embodiments, the 18F-radiolabeled prosthetic group or pharmaceutically acceptable salt is nonvolatile.

[00012] In some embodiments, the 18F-radiolabeled compound has the structure or a pharmaceutically acceptable salt thereof.

[00013] In certain embodiments, the pyridine ring of the 18F-labeled prosthetic group contains one or more additional substituents which do not interfere with fluorination of the molecule. In some embodiments, the additional substituent is a C 1_6 alkyl, for example, methyl, ethyl or propyl.

[00014] In a related aspect, provided herein is an 18F-radiolabeled prosthetic group having the structure wherein "OPEG" is [0(CH2)2]x, and x is an integer from 1 to 8, or a pharmaceutically acceptable salt thereof. In some embodiments, x is an integer from 2 to 6. In some embodiments, x is an integer from 3 to 5. In one embodiment, x is 4. In some embodiments, the 18F-radiolabeled prosthetic group or pharmaceutically acceptable salt is nonvolatile.

[00015] In certain aspects, provided herein is an 18F-radiolabeled protein-based probe comprising a 18F-radiolabeled prosthetic group as described herein, a bifunctional chelator or conjugating (BFC) moiety and a protein, wherein the radiolabeled protein-based probe has the following structure, õ ___________________________ [o(cH2)2k -- BFC I Protein or pharmaceutically acceptable salt thereof.

[00016] In certain aspects, provided herein is an 18F-radiolabeled protein-based probe comprising a 18F-radiolabeled prosthetic group as described herein, a bifunctional chelator or conjugating (BFC) and a protein, wherein the radiolabeled protein-based probe has the following structure, OPE BFC ¨ Protein or a pharmaceutically acceptable salt thereof.

[00017] In some embodiments, the BFC is a cyclooctyne comprising a reactive group that forms a covalent bond with an amine, carboxyl, carbonyl or thiol functional group on the protein. In some embodiments, the cyclooctyne is selected from the group consisting of dibenzocyclooctyne (DIBO), biarylazacyclooctynone (BARAC), dimethoxyazacyclooctyne (DIMAC) and dibenzocyclooctyne (DBCO). In some embodiments, the cyclooctyne is DBCO.

[00018] In some embodiments, the BFC further comprises a polyethylene glycol (PEG)y spacer arm, wherein y is an integer from 1 to 8. In some embodiments, y is an integer from 2 to 6. In some embodiments, y is 4 or 5.

[00019] In some embodiments, the BFC is DBCO-PEG4-NHS-Ester, DBCO-Su/fo-NHS-Ester, DBCO-PEG4-Acid, DBCO-PEG4-Amine or DBCO-PEG4-Maleimide. In some embodiments, the BFC is DBCO-PEG4-Maleimide.

[00020] In certain embodiments, provided herein is an 18F-radiolabeled protein-based probe with the following structure, F?rotetn ,6 J
o a NH
N1<) oJ
) J

wherein the maleimide group of the BFC is covalently linked to the thiol group on a cysteine residue of the protein. In some embodiments, the cysteine residue is at the C-terminus of the protein.

[00021] In related embodiments, the protein portion of the 18F-radiolabeled protein-based probe comprises a ligand. In some embodiments, the protein portion of the probe comprises an antibody or antibody fragment. In some embodiments, the protein portion of the probe comprises a fibronectin based scaffold (FBS).

[00022] In related embodiments, the protein portion of the 18F-radiolabeled protein-based probe binds to a biological molecule associated with a disease. In some embodiments, the disease is selected from the group consisting of solid cancers, hematopoietic cancers, hematological cancers, autoimmune disease, neurodegenerative disease, cardiovascular disease and pathogenic infections. In certain embodiments, the probe binds to a tumor-associated antigen. In certain embodiments, the probe binds to a protein present on a pathogenic organism, e.g., a virus, bacterium or fungus.

[00023] In certain embodiments, the 18F-radiolabeled protein-based probe provided herein may be in the form of a pharmaceutical composition.

[00024] In a related aspect, provided herein is a method of obtaining an image of an 18F-radiolabeled protein-based probe as provided herein, the method including the steps of (a) administering the 18F-radiolabeled protein-based probe to a subject; and (b) imaging in vivo the distribution of the 18F-radiolabeled protein-based probe by positron emission tomography (PET) scanning. In some embodiments, the imaged distribution of the 18F-radiolabeled protein-based probe is indicative of the presence or absence of a disease.

[00025] In a related aspect, provided herein is a method of diagnosing the presence of a disease in a subject, the method including the steps of (a) administering to a subject in need thereof an 18F-radiolabeled protein-based probe as provided herein which binds to a target molecule associated with the presence of the disease; and (b) obtaining an radio-image of at least a portion of the subject to detect the presence or absence of the 18F-radiolabeled protein-based probe; wherein the presence and location of the 18F-radiolabeled protein-based probe above background is indicative of the presence and location of the disease.

[00026] In a related aspect, provided herein is a method of monitoring the progress of a disease in a subject, the method including the steps of (a) administering to a subject in need thereof an 18F-radiolabeled protein-based probe as provided herein which binds to a target molecule associated with the presence of the disease at a first time point and obtaining an image of at least a portion of the subject to determine the amount of the diseased cells or tissue; and (b) administering to the subject the 18F-radiolabeled protein-based probe at one or more subsequent time points and obtaining an image of at least a portion of the subject at each time point; wherein the dimension and location of the diseased cells or tissue at each time point is indicative of the progress of the disease.

[00027] In a related aspect, provided herein is a method of quantifying diseased cells or tissues in a subject, the method including the steps of (a) administering to a subject having diseased cells or tissues with an 18F-radiolabeled protein-based probe as described herein which binds to a target molecule located with the diseased cells or tissues;
and (b) detecting radioactive emissions of the 18F in the diseased cells or tissue, wherein the level and distribution of the radioactive emissions in the diseased cells or tissues is a quantitative measure of the diseased cells or tissues.

[00028] In a related aspect, provided herein is a method of screening for an agent for treating a disease including the steps of (a) contacting a cells expressing a target protein associated with the disease with an 18F-radiolabeled protein-based probe as provided herein which binds to the target protein in the presence and absence of a candidate agent; and (b) imaging the cells in the presence and absence of the candidate agent using positron emission tomography (PET), wherein a decrease in the amount of radioactive emissions in the presence of the candidate agent is indicative of that the agent binds to the target protein.

[00029] In some embodiments of these methods, the disease is selected from the group consisting of solid cancers, hematopoietic cancers, hematological cancers, autoimmune disease, neurodegenerative disease, cardiovascular disease and pathogenic infection (e.g., viral, bacterial or fungal infections).

[00030] In a related aspect, provided herein is a method of obtaining a quantitative image of tissues or cells expressing a target protein, the method including the steps of contacting the cells or tissue with an 18F-radiolabeled protein-based probe as provided herein which binds to the target protein, and detecting or quantifying the tissue expressing the target protein using positron emission tomography (PET).

[00031] In some embodiments of the methods provided herein, the 18F-radiolabeled protein-based probe comprises a ligand. In some embodiments, the 18F-radiolabeled protein-based probe comprises an antibody or antibody fragment. In some embodiments, the 18F-radiolabeled protein-based probe comprises a fibronectin based scaffold (FBS).
In some embodiments, the 18F-radiolabeled protein-based probe binds to a tumor-associated antigen.
In still other embodiments, the 18F-radiolabeled protein-based probe binds to a protein present on a pathogenic organism (e.g., a virus, bacterium or fungus).

[00032] Also provided herein are kits containing the reaction precursors for producing the 18F-radiolabeled protein-based probes provided herein (e.g., a non-radiolabeled prosthetic group, BFC-linked protein probe and reagents for carrying out a bioorthogonal click reaction), and instructions for producing the 18F-radiolabeled protein-based probe.

[00033] Accordingly, in one aspect, provided herein is a method of producing a 18F-labeled prosthetic group having the following structure, e, 18F , riltru 1 1 Lwv,..,212jx¨Is13 N>1 wherein x is an integer from 1 to 8, the method including the steps of (a) providing a solution of a compound a with the following structure:

a =
R ............................... [0(CH2)21, -- N3 CH3 CH3 e 111() OTf wherein x is an integer from 1 to 8, and R is NO2, Br, F or and is ortho to the N atom of the pyridine ring; (b) providing a mixture of 18F in 180water, 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane and a weak base;
(c) drying the mixture from step (b) to form a solid; and (d) reacting the solution from step (a) with the solid from step (c) to form the 18F-labeled compound.

[00034] In certain embodiments, the method produces an 18F-pyridine prosthetic group with the following structure b N,/coco r0) m ..3) (where 18F is ortho to the N atom), and includes the steps of (a) providing a solution of the compound of the structure \ /

CH3 CH3 o OTf (where X is ortho to the N atom) where X is NO2, Br or (b) providing a mixture of 18F in 180water, 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane and weak base; (c) drying the mixture from step (b) to form a solid; and (d) reacting the solution from step (a) with the solid from step (c) to form the 18F-labeled compound.

[00035] In certain embodiments, the starting compound from step a) described above, X is NO2. In certain embodiments, X is attached to the carbon atom ortho to the N
atom and the PEG-N3 side chain. In certain embodiments the weak base in step b) is K2CO3, cesium carbonate or tetrabutylammonium hydroxide. In one embodiment, the weak base in step b) is K2CO3.

[00036] In a related aspect, a method for the 18F labeling of a protein is provided which includes the step of coupling the 18F-radiolabeled prosthetic group of formula b with the protein to be radiolabeled in a "click reaction" to form the 18F-radiolabeled protein. In some embodiments, the bioorthogonal click reaction is metal free (e.g., copper free click reaction).
In some embodiments, the protein comprises a bifunctional conjugating (BFC) moiety (e.g., a bifunctional chelator). In some embodiments, the protein comprises a cyclooctyne BFC. In some embodiments, the protein comprises DBCO-PEG4-Maleimide or is covalently linked to it.

[00037] Other features and advantages of the instant disclosure will be apparent from the following detailed description and examples, which should not be construed as limiting.
BRIEF DESCRIPTION OF THE DRAWINGS

[00038] Figure 1 is a schematic for the chemical synthesis of [18F]-E0 1-4PEG-DBCO-FPPEGA. The E01 portion of the molecule has the sequence set forth in SEQ ID
NO: 1.

[00039] Figure 2 is a bar graph depicting the tissue distribution of the 18F-A02 radiotracer in mice bearing bilateral PD-L1(+) L2987 and PD-L1(-) HT-29 xenografts as measured ex vivo by gamma counter.

[00040] Figure 3 is a composite image of 18F-E0 1 distribution in cynomologus monkeys.

[00041] Figure 4A and B depict images of in vitro autoradiography of 18F-A02 in xenograft and human lung tissues.

[00042] Figure 5 is a schematic for the production of [18F]-radiolabeled -A02-PEG-DBCO-FPPEGA using metal free "click chemistry".

[00043] Figure 6 is a schematic of the GE TRACERlab FX2 N Synthesis module for automated synthesis of [18F]-FPPEGA.

[00044] Figure 7 is a schematic of the Synthera Synthesis module (IBA) for automated synthesis of [18F] -FPPEGA.
DETAILED DESCRIPTION

[00045] Described herein are 18F- prosthetic groups and methods for producing the 18F-prosthetic groups. Also described herein are radiolabeled compositions containing the 18F-prosthetic groups and the use of these radiolabeled compositions to diagnose, localize, monitor and/or assess diseased cells and/or tissues, and related biological conditions.
Definitions

[00046] In order that the present description may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art, and conventional methods of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology are employed.

[00047] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. The use of "or" or "and" means "and/or" unless stated otherwise. Furthermore, use of the term "including" as well as other forms, such as "include", "includes", and "included", is not limiting.

[00048] As used herein, "about" means within plus or minus ten percent of a number. For example, "about 100" would refer to any number between 90 and 110.

[00049] As used herein, "medical imaging" refers to the techniques and processes used to create images of the subject's body (or parts thereof) for clinical purposes (medical procedures seeking to reveal, diagnose or monitor disease) or medical science (including the study of normal anatomy and physiology).

[00050] As used herein, "positron emission tomography" or "PET" refers to a non-invasive, nuclear medicine technique that produces a three-dimensional image of tracer location in the body. The method detects pairs of gamma rays emitted indirectly by a positron-emitting radionuclide (tracer), which is introduced into the body on a biologically active molecule.
PET imaging tools have a wide variety of uses and aid in drug development both preclinically and clinically. Exemplary applications include direct visualization of in vivo saturation of targets; monitoring uptake in normal tissues to anticipate toxicity or patient to patient variation; quantifying diseased tissue; tumor metastasis; and monitoring drug efficacy over time, or resistance over time.

[00051] The term "bioorthogonal chemistry" refers to any chemical reaction that can occur inside of living systems without interfering with native biochemical processes. The term includes chemical reactions that are chemical reactions that occur in vitro at physiological pH
in, or in the presence of water. To be considered bioorthogonol, the reactions are selective and avoid side-reactions with other functional groups found in the starting compounds. In addition, the resulting covalent bond between the reaction partners should be strong and chemically inert to biological reactions and should not affect the biological activity of the desired molecule.

[00052] The term "click chemistry" refers to a set of reliable and selective bioorthogonal reactions for the rapid synthesis of new compounds and combinatorial libraries. Properties of for click reactions include modularity, wideness in scope, high yielding, stereospecificity and simple product isolation (separation from inert by-products by non-chromatographic methods) to produce compounds that are stable under physiological conditions.
In radiochemistry and radiopharmacy, click chemisty is a generic term for a set of labeling reactions which make use of selective and modular building blocks and enable chemoselective ligations to radiolabel biologically relevant compounds in the absence of catalysts. A "click reaction" can be with copper, or it can be a copper-free click reaction.

[00053] The term "prosthetic group" or "bifunctional labeling agent" refers to a small organic molecule containing a radionulide (e.g., 18F) that is capable of being linked to peptides or proteins.

[00054] The term "chelator ligand" as used herein with respect to radiopharmaceutical chemistry refers to a bifunctional chelator or bifunctional conjugating (BFC) moiety (used interchangeably herein) that covalently links a radiolabeled prosthetic group to a biologically active targeting molecule (e.g., peptide or protein). BFCs utilize functional groups such as carboxylic acids or activated esters for amide couplings, isothiocyanates for thiourea couplings and maleimides for thiol couplings.

[00055] As used herein, "target" as a general reference to a "biological target" refers to a cell, tissue (e.g., cancer or tumor), a pathogenic microorganism (e.g., bacteria, virus, fungus, plant, prion, protozoa or portion thereof) or other molecule associated with a biological pathway, or a biological phenomenon, such as tissue inflammation, plaque formation, etc.

[00056] The term "targeting ligand", "targeting agent" or "targeting molecule"
are used interchangeably to refer to a molecule, such as peptide, protein, glycoprotein, etc., that binds to another molecule. In certain embodiments, a targeting agent is bound to the 18F-prosthetic group in order to "target" a molecule associated with a particular cell, tissue, pathogen or biological pathway.

[00057] "Polypeptide" as used herein refers to any sequence of two or more amino acids, regardless of length, post-translation modification, or function.
"Polypeptide," "peptide," and "protein" are used interchangeably herein. Polypeptides can include natural amino acids and non-natural amino acids such as those described in U.S. Pat. No. 6,559,126, incorporated herein by reference. Polypeptides can also be modified in any of a variety of standard chemical ways (e.g., an amino acid can be modified with a protecting group;
the carboxy-terminal amino acid can be made into a terminal amide group; the amino-terminal residue can be modified with groups to, e.g., enhance lipophilicity; or the polypeptide can be chemically glycosylated or otherwise modified to increase stability or in vivo half-life). Polypeptide modifications can include the attachment of another structure such as a cyclic compound or other molecule to the polypeptide and can also include polypeptides that contain one or more amino acids in an altered configuration (i.e., R or S; or, L or D).

[00058] An "isolated" polypeptide is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the polypeptide will be purified (1) to greater than 95% by weight of polypeptide as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing condition using Coomassie blue or, preferably, silver stain. Isolated polypeptide includes the polypeptide in situ within recombinant cells since at least one component of the polypeptide's natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.

[00059] The terms "specifically binds," "specific binding," "selective binding, and "selectively binds," as used interchangeably herein refers to an peptide or polypeptide that exhibits affinity for a biological target, but does not significantly bind (e.g., less than about 10% binding) to a other molecules as measured by a technique available in the art such as, but not limited to, Scatchard analysis and/or competitive binding assays (e.g., competition ELISA, BIACORE assay).

[00060] The term "preferentially binds" as used herein refers to the situation in which an peptide or protein binds a selected biological target at least about 20%
greater than it binds a different biological target as measured by a technique available in the art such as, but not limited to, Scatchard analysis and/or competitive binding assays (e.g., competition ELISA, BIACORE assay).

[00061] The term "KD," as used herein, is intended to refer to the dissociation equilibrium constant of a protein- protein interaction (e.g., 18F-radiolabeled-protein based probe-target molecule) or the affinity of an 18F-radiolabeled-protein based probe for a target protein, as measured using a surface plasmon resonance assay or a cell binding assay. A
"desired KD,"
as used herein, refers to a KD of an 18F-radiolabeled-protein based probe that is sufficient for the purposes contemplated. For example, a desired KD may refer to the KD of an radiolabeled-protein based probe required to elicit a functional effect in an in vivo imaging assay or in vitro assay, e.g., a cell-based luciferase assay.

[00062] The term "kaõ", as used herein, is intended to refer to the association rate constant for the association of an 18F-radiolabeled-protein based probe into an 18F-radiolabeled-protein based probe/target protein complex.

[00063] The term "kd", as used herein, is intended to refer to the dissociation rate constant for the dissociation of an 18F-radiolabeled-protein based probe from the 18F-radiolabeled-protein based probe/target protein complex.

[00064] The term "IC50", as used herein, refers to the concentration of an 18F-radiolabeled-protein based probe that inhibits a response, either in an in vitro or an in vivo assay, to a level that is 50% of the maximal inhibitory response, i.e., halfway between the maximal inhibitory response and the untreated response.

[00065] The term "PK" is an acronym for "pharmacokinetic" and encompasses properties of a compound including, by way of example, absorption, distribution, metabolism, and elimination by a subject. A "PK modulation protein" or "PK moiety" as used herein refers to any protein, peptide, or moiety that affects the pharmacokinetic properties of a biologically active molecule when fused to or administered together with the biologically active molecule.
Examples of a PK modulation protein or PK moiety include PEG, human serum albumin (HSA) binders (as disclosed in U.S. Publication Nos. 2005/0287153 and 2007/0003549, PCT
Publication Nos. WO 2009/083804 and WO 2009/133208), human serum albumin and variants thereof, transferrin and variants thereof, Fc or Fc fragments and variants thereof, and sugars (e.g., sialic acid).

[00066] The "serum half-life" of a protein or compound can generally be defined as the time taken for the serum concentration of the polypeptide to be reduced by 50%, in vivo, for example due to degradation of the sequence or compound and/or clearance or sequestration of the sequence or compound by natural mechanisms. The half-life can be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be clear to the person skilled in the art, and may for example generally involve the steps of suitably administering to a subject a suitable dose of the amino acid sequence or compound described herein; collecting blood samples or other samples from the subject at regular intervals;
determining the level or concentration of the amino acid sequence or compound described herein in said blood sample; and calculating, from (a plot of) the data thus obtained, the time until the level or concentration of the amino acid sequence or compound described herein has been reduced by 50% compared to the initial level upon dosing. Reference is, for example, made to the standard handbooks, such as Kenneth, A. et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and in Peters et al., Pharmacokinete Analysis:
A Practical Approach (1996). Reference is also made to Gibaldi, M. et al., Pharmacokinetics, 2nd Rev. Edition, Marcel Dekker (1982).

[00067] Half-life can be expressed using parameters such as the ti/2-alpha, t112-beta, HL_Lambda_z, and the area under the curve (AUC). In the present specification, an "increase in half-life" refers to an increase in any one of these parameters, any two of these parameters, any three of these parameters or all four of these parameters. An "increase in half-life" in particular refers to an increase in the t112-beta, and/or HL_Lambda_z, either with or without an increase in the ti/2-alpha and/or the AUC or both.

[00068] As used herein, the term "linked" refers to the association of two or more molecules.
The linkage can be covalent or non-covalent. The linkage also can be genetic (i.e., recombinantly fused). Such linkages can be achieved using a wide variety of art recognized techniques, such as chemical conjugation and recombinant protein production.

[00069] The terms "diagnosis" or "detection" can be used interchangeably.
Whereas diagnosis usually refers to defining a tissue's specific histological status, detection recognizes and locates a tissue, lesion or organism containing a particular detectable target.

[00070] The term "detectable" refers to the ability to detect a signal over the background signal. The term "detectable signal" as used herein in the context of imaging agents and diagnostics, is a signal derived from non-invasive imaging techniques such as, but not limited to, positron emission tomography (PET). The detectable signal is detectable and distinguishable from other background signals that may be generated from the subject. In other words, there is a measurable and statistically significant difference (e.g., a statistically significant difference is enough of a difference to distinguish among the detectable signal and the background, such as about 0.1%, 1%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, or 40% or more difference between the detectable signal and the background) between the detectable signal and the background. Standards and/or calibration curves can be used to determine the relative intensity of the detectable signal and/or the background.

[00071] A "detectably effective amount" of a composition comprising an imaging agent described herein is defined as an amount sufficient to yield an acceptable image using equipment that is available for clinical use. A detectably effective amount of an imaging agent provided herein may be administered in more than one injection. The detectably effective amount can vary according to factors such as the degree of susceptibility of the individual, the age, sex, and weight of the individual, idiosyncratic responses of the individual, and the like. Detectably effective amounts of imaging compositions can also vary according to instrument and methodologies used. Optimization of such factors is well within the level of skill in the art.

[00072] As used herein, "administering," as used in the context of imaging agents refers to the physical introduction of a composition comprising an imaging agent to a subject, using any of the various methods and delivery systems known to those skilled in the art. Preferred routes of administration for the imaging agents described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration"
as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. Alternatively, an imaging agent described herein can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.

[00073] The terms "co-administration" or the like, as used herein, are meant to encompass administration of the selected pharmaceutical agents to a single patient, and are intended to include regimens in which the agents are administered by the same or different route of administration or at the same or different time.

[00074] The terms "patient" and "subject" refer to any human or non-human animal that receives a composition comprising an imaging agent described herein. For in vitro applications, such as in vitro diagnostic and research applications, body fluids and cell samples of the above subjects will be suitable for use, such as blood, urine, or tissue samples.

[00075] The term "sample" can refer to a tissue sample, cell sample, a fluid sample, and the like. The sample may be taken from a subject. The tissue sample can include hair (including roots), buccal swabs, blood, saliva, semen, muscle, or from any internal organs. The fluid may be, but is not limited to, urine, blood, ascites, pleural fluid, spinal fluid, and the like. The body tissue can include, but is not limited to, skin, muscle, endometrial, uterine, and cervical tissue.

[00076] The term "isotopically pure" means that the element, compound, or composition contains a greater proportion of one isotope in relation to other isotopes. In certain embodiments, the element, compound, or composition is greater than about 40%, 50%, or 60% isotopically pure.

[00077] As used herein, a labeled molecule is "purified" when the labeled molecule is partially or wholly separated from unlabeled molecules, so that the fraction of labeled molecules is enriched compared to the starting mixture. A "purified" labeled molecule may comprise a mixture of labeled and unlabeled molecules in almost any ratio, including but not limited to about 5:95; 10:90; 15:85; 20:80; 25:75; 30:70; 40:60; 50:50; 60:40;
70:30; 75:25;
80:20; 85:15; 90:10; 95:5; 97:3; 98:2; 99:1 or 100:0.

[00078] The group "OTf" refers to triflate having the formula CF3S03 or trifluoromethanesulfate.

[00079] The term "halo" or, alternatively, "halogen" or "halide" means fluoro, chloro, bromo or iodo.

[00080] Throughout the specification, groups and substituents thereof may be chosen by one skilled in the field to provide stable moieties and compounds and compounds useful as pharmaceutically-acceptable compounds and/or intermediate compounds useful in making pharmaceutically-acceptable compounds.

[00081] Various aspects described herein are described in further detail in the following subsections.
18F Radiolabeled Prosthetic Groups

[00082] In one aspect, provided herein is an 18F-radiolabeled compound containing a prosthetic group for use in a bioorthogonal reaction involving 1,3-dipolar cycloaddition between an azide and a cyclooctyne which proceeds selectively under water tolerant conditions. The 18F-radiolabeled prosthetic groups of the invention are soluble in 100%
aqueous, and there is no need for an organic phase to link the prosthetic group to a peptide or protein molecule. This feature is particularly advantageous as many biologics (e.g., peptides or proteins), cannot withstand even small amounts of organic solvents, with degradation and aggregation issues.

[00083] Additionally, unlike aliphatic prosthetic groups, with the instant prosthetic group, the 18F fluorination reaction can be monitored with UV. The 18F-radiolabeled prosthetic groups described herein are not volatile. Moreover, 18F-radiolabeled prosthetic groups provided may be incorporated into biologics using a copper free click chemisty, e.g., as described in the Examples, thus avoiding the stability issues observed in some biologics when copper mediated click chemistry is used.

[00084] In one aspect, provided herein is a PEGylated 18F-pyridine covalently bound to an azide with the following structure, 18F_ rsu Ls-eµson 12,2jx-143 or a pharmaceutically acceptable salt thereof, wherein x is an integer from 1 to 8. In some embodiments, x is an integer from 2 to 6. In some embodiments x is an integer from 3 to 5.
In some embodiments, x is 4. In related embodiments, 18F is attached to the pyridine ortho to the N atom. In some embodiments, the [0(CH2)2]õ moiety is present in the 1-3 configuration relative to the nitrogen on the pyridine ring. In some embodiments, the [0(CH2)2]x moiety is present in the 1-2 configuration relative to the nitrogen on the pyridine ring. In other embodiments, the [0(CH2)2]x moiety is present in the 1-4 configuration relative to the nitrogen on the pyridine ring.

[00085] In some embodiments, the 18F-radiolabeled compound has the structure ¨[0(CF12)2lx¨N3 N>
or a pharmaceutically acceptable salt thereof, wherein x is an integer from 1 to 8. In some embodiments, x is an integer from 2 to 6. In some embodiments x is an integer from 3 to 5.
In some embodiments, x is 4. In some embodiments, the [0(CH2)2]x moiety is present in the 1-3 configuration relative to the nitrogen on the pyridine ring. In some embodiments, the [0(CH2)2]x moiety is present in the 1-2 configuration relative to the nitrogen on the pyridine ring. In other embodiments, the [0(CH2)2]x moiety is present in the 1-4 configuration relative to the nitrogen on the pyridine ring.

[00086] In some embodiments, the 18F-radiolabeled compound has the structure NI
[0(CH2)2]¨N3 wherein x is an integer from 1 to 8. In some embodiments, x is an integer from 2 to 6. In some embodiments x is an integer from 3 to 5. In some embodiments, x is 4.

[00087] In some embodiments, the 18F-radiolabeled compound is [18F]-3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine (18F-FPPEGA) and has the structure 18F =

[00088] In alternative embodiments, the 18F-radiolabeled prosthetic group may contain additional groups on the pyridine ring which do not interfere with the fluorination reaction.
In certain embodiments, additions to the pyridine ring include C1_6 alkyl groups, for example methyl, ethyl and propyl.

[00089] In still other embodiments, the 18F-radiolabeled prosthetic group is a fused ring system with the following structure:
N

wherein "OPEG" is [0(CH2)2]õ, and x is an integer from 1 to 8. In some embodiments, x is an integer from 2 to 6. In some embodiments x is an integer from 3 to 5. In some embodiments, x is 4.

[00090] In a related aspect, provided herein is a method of preparing a PEGylated 18F-pyridine covalently bound to an azide with the following structure, 18F_ rrtirsu wherein x is an integer from 1 to 8, the method comprising the steps of (a) providing a solution of a compound a with the following structure:
a ------------------------------ rrter.0 p..4k=....212ix ------------------------- N3 N

0 Tf wherein x is an integer from 1 to 8, and R is NO2, Br, F or and is ortho to the N atom of the pyridine ring;
(b) providing a mixture of 18F in 180water, 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane and a weak base;
(c) drying the mixture from step (b) to form a solid; and (d) reacting the solution from step (a) with the solid from step (c) to form the 18F-labeled compound.

[00091] In certain embodiments, the method produces a 18F-pyridine prosthetic group with the following structure b ro) ro N3 ) (where 18F is ortho to the N atom), and includes the steps of (a) providing a solution of the compound of the structure 0-r j-0 OTf (where X is ortho to the N atom) where X is NO2, Br or =
(b) providing a mixture of 18F in 180water, 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane and weak base, such as K2CO3;
(c) drying the mixture from step (b) to form a solid; and (d) reacting the solution from step (a) with the solid from step (c) to form the 18F-labeled compound.

[00092] In certain embodiments, the method further comprises the step of producing a compound with the following structure a R ....................................... [0(CH2)21, N3 according to the Scheme I shown below:

Ts0 N3 , Hi::: 0 0 Na 0 N=N+N- XN
lim.. -Now 0 0 Et0H, reflux 0 NaH, DMF

32%

Ts0 Ts 0 X-._o N ¨
X= Br, NO2, F

[00093] In certain embodiments, the method comprises producing the 18F-pyridine prosthetic group [18F]-3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine (18F-FPPEGA), e, from_d, according to the following reaction conditions:
K.2.2.2 K2CO3 N e-C) _______________________________________ w X r0) DMSO 18F ro) N3)( 1 0 mins N3(0) d e ([189-FPPEGA) II. 18F-radiolabeled Biological Probes

[00094] In certain aspects, provided herein are 18F-radiolabeled probes or agents with the following structure, ____________________________ [0(cH2)2], -- BFC -11 Protein 14, wherein x is an integer from 1 to 8. In some embodiments, x is an integer from 2 to 6. In some embodiments x is an integer from 3 to 5. In some embodiments, x is 4.
BFC

[00095] Bifunctional chelators or conjugating (BFC) moieties, which can be used in the radiolabeled compositions disclosed herein, are commercially available (e.g., Sigma Aldrich;
Click Chemistry Tools), or may be synthesized according to well-known chemical reactions.

[00096] In certain embodiments, the BFC is selected from cyclooctyne based agents (e.g., DBCO, DIBO), DFO, DOTA and its derivatives (CB-D02A, 3p-C-DEPA, TCMC, Oxo-DO3A), TE2A, CB-TE2A, CB-TE1A1P, CB-TE2P, MM-TE2A, DM-TE2A, diamsar and derivatives, NODASA, NODAGA, NOTA, NETA, TACN-TM, DTPA, 1B4M-DTPA, CHX-A"-DTPA, TRAP (PRP9), NOPO, AAZTA and derivatives (DATA), H2dedpa, H4octapa, H2azapa, H5decapa, H6phospa, HBED, SHBED, BPCA, CP256, PCTA, HEHA, PEPA, EDTA, TETA, and TRITA based chelating agents, and close analogs and derivatives thereof.
Suitable combinations of chelating agents and radionuclides are extensively described in Price et al., Chem Soc Rev 2014;43:260-90.

[00097] In certain embodiments, the BFC is a cyclooctyne comprising a reactive group that forms a covalent bond with an amine, carboxyl, carbonyl or thiol functional group on the targeting protein or peptide. Reactive groups on the cyclooctyne include esters, acids, hydroxyl groups, aminooxy groups, maliemides, a-halogenketones and a-halogenacetamides.

[00098] In certain embodiments, the BFC is a cyclooctyne, such as dibenzocyclooctyne (DIBO), biarylazacyclooctynone (BARAC), dimethoxyazacyclooctyne (DIMAC) and dibenzocyclooctyne (DBCO). In certain embodiments, the cyclootyne is DBCO.

[00099] In certain embodiments, the cyclooctyne comprises a hydrophilic polyethylene glycol (PEG)y spacer arm, wherein y is an integer from 1 to 8. In certain embodiments, y is an integer from 2 to 6. In certain embodiments, y is 4 or 5.

[000100] In certain embodiments, the BFC is DBCO-PEG4-NHS-Ester or DBCO-Sulfo-NHS-Ester which react specifically and efficiently with a primary amine (e.g., side chain of lysine residues or aminosilane-coated surfaces). In certain embodiments, the BFC is DBCO-PEG4-Acid with terminal carboxylic acid (¨COOH) that can be reacted with primary or secondary amine groups in the presence activators (e.g. EDC) forming a stable amide bond.
In certain embodiments, the BFC is DBCO-PEG4-Amine which reacts with carboxyl groups in the presence of activators (e.g. EDC, or DCC) or with activated esters (e.g. NHS esters) forming stable amide bonds.

[000101] In certain embodiments, the BFC is DBCO-PEG4-Maleimide which reacts with sulfhydryl groups on cysteine residues, e.g., cysteine residues near the C-terminus of the polypeptide.

[000102] In certain embodiments, for labeling a protein, the protein is first modified to incorporate a cysteine for attaching the prosthetic group. For example, a cysteine may be added to the C-terminus of the protein. In certain embodiments, PxCy, wherein P is proline, C is cysteine, x is an integer that is at least 0 (e.g., 0, 1 or 2) and y is an integer that is at least 1, is added to the C-terminus of the protein. Methods for making modifications to proteins are well-known in the art.

[000103] In certain embodiments, the 18F-radiolabeled probe or agent has the following structure a, Pro!en '0 (.6 H
a N N '0 N ) N' o \9 wherein, the BFC is conjugated to the protein at a cysteine residue.

[000104] The 18F-radiolabeled targeting agents described herein may be produced using bioorthogonal, metal free click chemistry in medium suitable for direct use in vivo (e.g., saline) according to the procedures described herein.
III. Protein/Peptide Targeting Molecules

[000105] The 18F-prosthetic groups provided herein may be attached to virtually any targeting molecule, so long as it contains a derivatizable group that may be modified without affecting the interaction between the targeting molecule and the in vivo biological target (e.g., cell or tissue).

[000106] In some embodiments, the targeting molecule is a peptide or protein, including, but not limited to, antibodies, antibody fragments, fibronectin based molecules and ligands (e.g., hormones, growth factors, cytokines, chemokines, interleukins and angiogenic factors). In some embodiments, the targeting molecule will comprise one or more binding sites for a target associated with a disease or condition, such as a tumor associated or autoimmune antigen, or a protein displayed by a pathogenic organism such as a virus, bacterium, fungus or protozoan.

[000107] In some embodiments, the 18F-labeled peptides or protein may be selected to bind directly to a targeted cell, tissue, pathogenic organism or other target for imaging and/or detection. In other embodiments, 18F labeled protein or peptide may be selected to bind directly or indirectly to the in vivo target molecule. For example, a first protein or peptide may administered to the subject, followed by a second 18F-labeled molecule which binds to the first.
Peptides

[000108] Peptides having as few as two amino acid residues, preferably two to ten residues, may be used and may also be coupled to other moieties. The targetable construct may also comprise unnatural amino acids, e.g., D-amino acids, in the backbone structure to increase the stability of the peptide in vivo. In alternative embodiments, other backbone structures such as those constructed from non-natural amino acids or peptoids may be used.

[000109] In some embodiments, peptides which may be used include ligands, peptide vaccines, and epitopes. The peptides used as targetable constructs are conveniently synthesized on an automated peptide synthesizer using a solid-phase support and standard techniques of repetitive orthogonal deprotection and coupling. N-terminal residues may be acetylated to increase serum stability. Such protecting groups will be known to the skilled artisan. See Greene and Wuts Protective Groups in Organic Synthesis, 1999 (John Wiley and Sons, N.Y.).
Antibodies

[000110] In certain embodiments, the targeting molecule used in the radiotracer composition described herein is an antibody. The term "antibody" as used to herein may include whole antibodies and any antigen binding fragments (i.e., "antigen-binding portions") or single chains thereof. By way of example "antibody" may refer to both naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and nonhuman antibodies; bispecific antibodies;
wholly synthetic antibodies; and single chain antibodies. As used herein, the term "antigen" refers to any natural or synthetic immunogenic substance, such as a protein, peptide, or hapten.

[000111] The targeting molecules described herein may incorporate any antibody or fragment known in the art that has binding specificity for a target antigen associated with a disease state or condition. Antibodies useful as targeting molecules may be commercially obtained from a wide variety of sources (e.g., ATTC, Manassas, VA), and/or have published variable region sequences which may be produced according to art recognized recombinant techniques. In some embodiments, exemplary antibodies for use in the present methods include an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PDL-1 antibody, an anti-0X40 (also known as CD134, TNFRSF4, ACT35 and/or TXGP1L) antibody, or an anti-LAG-3 antibody.

[000112] Antibodies used in the compositions and methods described herein can be produced using a variety of known techniques. Immunization protocols and techniques for isolation of immunized splenocytes are well established in the art. The production of monoclonal antibodies using the standard somatic cell hybridization technique described by Kohler and Milstein, Nature 256: 495 (1975), as well as viral or oncogenic transformation of B
lymphocytes, phage display technique using libraries of human antibody genes are also routine. In addition, standard methodologies for the production of chimeric and humanized antibodies are readily available (see e.g., U.S. Patent No. 4,816,567 to Cabilly et al.;U.S.
Patent No. 5,225,539 to Winter, and U.S. Patent Nos. 5,530,101; 5,585,089;
5,693,762 and 6,180,370 to Queen et al.).

[000113] In certain embodiments, the targeting molecule used in the radiotracer composition is an antigen binding fragment. As used herein, the term "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VII, CL and CH1 domains; (ii) a F(abt)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL
and VH
domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) or (vii) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules known as single chain Fv (scFv);
see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad.
Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These and other potential constructs are described at Chan & Carter (2010) Nat. Rev. Immunol. 10:301.
These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
Antigen-binding portions can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

[000114] In certain embodiments, the antibody used is modified to modulate, e.g., decrease the half-life of the antibody or rapid clearance for use in the medical imaging methods described herein. Modifications such as I253A (Hornick et al. (2000) J. Nucl.
Med. 41:355) and H435A/R I253A or H310A (Kim et al. (2000) Eur. J. Immunol. 29:2819) in Fc of human IgG1 can decrease FcRn binding. See also Kenanova et al. (2005) Cancer Res.
65:622.
Other means to enhance clearance include formatting the antigen binding domains of the present invention as antibody fragments lacking the ability to bind FcRn, such as Fab fragments. Such modification can reduce the circulating half-life of an antibody from a couple of weeks to a matter of hours. Selective PEGylation of antibody fragments can then be used to fine-tune (increase in increments) the half-life of the antibody fragments if necessary. Chapman et al. (1999) Nat. Biotechnol. 17:780.

[000115] Radiotracer compositions containing an antibody or antigen binding fragment thereof can be assayed for retention of binding specificity in vitro and/or in vivo. Methods for analyzing binding affinity, cross-reactivity, and binding kinetics of various antibody compositions include standard assays known in the art, for example, ELISA, Western Blotting, flow cytometry, and BIACORE surface plasmon resonance (SPR) analysis using a BIACORE 2000 SPR instrument (Biacore AB, Uppsala, Sweden).

[000116] Exemplary proteins for use in the radiolabeled probes described herein include any known antibody or alternative scaffold protein, such as Adnectins, that specifically binds to a target, and does significantly cross-react with unrelated targets.
Fibronectin Based Protein (FBS)

[000117] In some embodiments, the targeting molecule used in the radiotracer compositions described herein is a FBS protein. Generally, FBS protein molecules have inherently rapid blood clearance rates, which can be advantageous for use with 18F in imaging technologies by minimizing the amount of time needed for background probe signals from non-relevant tissue. Rapid clearing probers allow high contrast images to be collected the same day the probe is injected, and very importantly, can also serve to reduce overall radiation exposure to the subject.

[000118] As used herein, a "fibronectin based scaffold" or "FBS" protein or moiety refers to proteins or moieties that are based on a fibronectin type III ("Fn3") repeat.
Fn3 is a small (about 10 kDa) domain that has the structure of an immunoglobulin (Ig) fold (i.e., an Ig-like I3-sandwich structure, consisting of seven I3-strands and six loops).
Fibronectin has 18 Fn3 repeats, and while the sequence homology between the repeats is low, they all share a high similarity in tertiary structure. Fn3 domains are also present in many proteins other than fibronectin, such as adhesion molecules, cell surface molecules, e.g., cytokine receptors, and carbohydrate binding domains. For reviews see Bork et al., Proc. Natl. Acad.
Sci. USA, 89(19):8990-8994 (1992); Bork et al., J. Mol. Biol., 242(4):309-320 (1994);
Campbell et al., Structure, 2(5):333-337 (1994); Harpez et al., J. Mol. Biol., 238(4):528-539 (1994)). The term "FBS" protein or moiety is intended to include scaffolds based on Fn3 domains from these other proteins (i.e., non fibronectin molecules).

[000119] An Fn3 domain is small, monomeric, soluble, and stable. It lacks disulfide bonds and, therefore, is stable under reducing conditions. Fn3 domains comprise, in order from N-terminus to C-terminus, a beta or beta-like strand, A; a loop, AB; a beta or beta-like strand, B; a loop, BC; a beta or beta-like strand, C; a loop, CD; a beta or beta-like strand, D; a loop, DE; a beta or beta-like strand, E; a loop, EF; a beta or beta-like strand, F;
a loop, FG; and a beta or beta-like strand, G. The seven antiparallel I3-strands are arranged as two beta sheets that form a stable core, while creating two "faces" composed of the loops that connect the beta or beta-like strands. Loops AB, CD, and EF are located at one face ("the south pole") and loops BC, DE, and FG are located on the opposing face ("the north pole").
There are at least 15 different Fn3 modules in human Fibronectin, and while the sequence homology between the modules is low, they all share a high similarity in tertiary structure.

[000120] The loops in Fn3 molecules are structurally similar to complementary determining regions (CDRs) of antibodies, and when altered, may be involved in binding of the Fn3 molecule to a target, e.g., a target protein. Other regions of Fn3 molecules, such as the beta or beta-like strands and N-terminal or C-terminal regions, when altered, may also be involved in binding to a target. Any or all of loops AB, BC, CD, DE, EF and FG may participate in binding to a target. Any of the beta or beta-like strands may be involved in binding to a target. Fn3 domains may also bind to a target through one or more loops and one or more beta or beta-like strands. Binding may also require the N-terminal or C-terminal regions. An FBS domain for use in a protein may comprise all loops, all beta or beta-like strands, or only a portion of them, wherein certain loops and/or beta or beta-like strands and/or N- or C-terminal regions are modified (or altered), provided that the FBS domain preferably binds specifically to a target. For example, an FBS domain may comprise 1, 2, 3, 4, 5 or 6 loops, 1, 2, 3, 4, 5, 6, 7, or 8 beta strands, and optionally an N-terminal and/or C-terminal region, wherein one or more loops, one or more beta strands, the N-terminal region and/or the C-terminal regions are modified relative to the wild-type FBS domain.

[000121] An example of FBS proteins that are based on human 10Fn3 domains are adnectins (Adnexus, a wholly owned subsidiary of Bristol-Myers Squibb). Adnectins are 10Fn3 molecules in which CDR-like loop regions, I3-strands, N-terminal and/or C-terminal regions of a 10Fn3 domain has been modified to evolve a protein capable of binding to a compound of interest. For example, U.S. Patent No. 7,115,396 describes 10Fn3 domain proteins wherein alterations to the BC, DE, and FG loops result in high affinity TNFcc binders.
U.S. Patent No.
7,858,739 describes Fn3 domain proteins wherein alterations to the BC, DE, and FG loops result in high affinity VEGFR2 binders.

[000122] In certain embodiments, an FBS moiety is based on an Fn3 repeat other than the 10th repeat of the type III domain of fibronectin, e.g., human fibronectin.
For example, an , FBS moiety may be similar to any of the other fibronectin type III repeats, e.g., the 1st, 2nd 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, and 18th Fn3 repeats. In yet other embodiments, an FBS moiety may be from a molecule other than fibronectin.
Exemplary FBS moieties may be derived from tenascin, a protein that is composed of 15 Fn3 domains with similar sequence similarities to one another as found in fibronectin. These repeats are described, e.g., in Jacobs et al., Protein Engineering, Design &
Selection, 25:107 (2012). Based on the homology of the repeats in the fibronectin molecule and those in the tenascin molecule, artificial molecules based on these homologies have been created.
Proteins comprising a consensus amino acid sequence based on the homology of the domains in the fibronectin molecule are referred to as Fibcon and FibconB (WO
2010/093627 and Jacobs et al. (2012) supra.) and those based on the homology of the domains in the tenascin molecule are referred to as Tencon (WO 2010/051274, WO 2010/051310 and WO
2011/137319, which are specifically incorporated by reference herein). A
Fibcon, FibconB
or Tencon moiety, or target binding variants thereof, whether by istelf or linked to a heterologous moiety may be fused as described herein. Fn3 domains from other proteins, e.g., cell surface hormone and cytokine receptors, chaperonins, and carbohydrate-binding domains, may be conjugated as described herein.

[000123] FBS proteins specific for any desired target molecule can be generated and tested using art recognized methods. Methods for testing the binding properties of FBS proteins are also well-known. For example, one way to rapidly make and test Fn3 domains with specific binding properties is the nucleic acid-protein fusion technology of Adnexus, a Bristol-Myers Squibb R&D Company. This disclosure utilizes the in vitro expression and tagging technology, termed TROfusion' which exploits nucleic acid-protein fusions (RNA-and DNA-protein fusions) to identify novel polypeptides and amino acid motifs that are important for binding to proteins. Nucleic acid-protein fusion technology is a technology that covalently couples a protein to its encoding genetic information. For a detailed description of the RNA-protein fusion technology and fibronectin-based scaffold protein library screening methods see Szostak et al., U.S. Pat. Nos. 6,258,558, 6,261,804, 6,214,553, 6,281,344, 6,207,446, 6,518,018 and 6,818,418; Roberts et al., Proc. Natl. Acad. Sci., 1997;94:12297-12302; and Kurz et al., Molecules, 2000;5:1259-64, all of which are herein incorporated by reference.

[000124] Exemplary FBS proteins or moieties included, but are not limited to those which bind to mesothelian, glypican, TL1A, CD8, myostatin, LPA1 receptors, TNF-alpha, VEGFR2, PCSK9, IL-23, EGFR or IGF1R and those which are described, e.g., in WO

2010/093627, WO 2011/130324, WO 2009/083804, WO 2009/133208, WO 02/04523, WO

2012/016245, WO 2009/023184, WO 2010/051310, WO 2011/020033, WO 2011/051333, WO 2011/051466, WO 2011/092233, WO 2011/100700, WO 2011/130324, WO
2011/130328, WO 2011/137319, WO 2010/051274, WO 2009/086116, WO 09/058379, W02013/067029 and W02012/016245 (all of which are specifically incorporated by reference herein): any of the FBS proteins or moieties described in these publications may be used as described herein.

[000125] In some embodiments, the FBS protein binds to PDL-1. In some embodiments, the FBS protein comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[000126] In certain embodiments, an imaging agent, e.g., comprising an FBS
protein, is linked to a moiety that modulates, e.g., increases, its blood PK by small increments to enhance the imaging contrast or increase avidity of the 18F-labeled targeting agent. In some embodiments, the clearance rate of the polypeptide in a mammal (e.g., mouse, rat, or human) is, or is increased by greater than two-fold, greater than three-fold, greater than four-fold or greater than five-fold relative to the unmodified FBS protein. Moieties that slow clearance of a protein from the blood, herein referred to as "PK moieties", include polyoxyalkylene moieties (e.g., polyethylene glycol), sugars (e.g., sialic acid), and well-tolerated protein moieties (e.g., Fc and fragments and variants thereof, transferrin, or serum albumin). The FBS protein may also be fused to albumin or a fragment (portion) or variant of albumin as described in U.S. Publication No. 2007/0048282, or may be fused to one or more serum albumin binding FBS proteins, as described herein.

[000127] Other PK moieties that can be used in the invention include those described in Kontermann et al., (Current Opinion in Biotechnology 2011;22:868-76), herein incorporated by reference. Such PK moieties include, but are not limited to, human serum albumin fusions, human serum albumin conjugates, human serum albumin binders (e.g., Adnectin PKE, AlbudAb, ABD), XTEN fusions, PAS fusions (i.e., recombinant PEG mimetics based on the three amino acids proline, alanine, and serine), carbohydrate conjugates (e.g., hydroxyethyl starch (HES)), glycosylation, polysialic acid conjugates, and fatty acid conjugates.

[000128] In some embodiments, the invention provides 18F-labeled FBS proteins fused to a PK moiety that is a polymeric sugar. In some embodiments, the PK moiety is a polyethylene glycol moiety. PEG is a well-known, water soluble polymer that is commercially available or can be prepared by ring-opening polymerization of ethylene glycol according to methods well known in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161).

[000129] The term "PEG" is used broadly to encompass any polyethylene glycol molecule, without regard to size or to modification at an end of the PEG, and can be represented by the formula: X-0(CH2CH20)._1CH2CH20H, where n is 2 or more, e.g., 20 to 2300 and X
is H
or a terminal modification, e.g., a C 1_4 alkyl. PEG can contain further chemical groups which are necessary for binding reactions, which result from the chemical synthesis of the molecule;
or which act as a spacer for optimal distance of parts of the molecule. In addition, such a PEG
can consist of one or more PEG side-chains which are linked together. PEGs with more than one PEG chain are called multiarmed or branched PEGs. Branched PEGs are described in, for example, European Published Application No. 473084A and U.S. Pat. No.
5,932,462.

[000130] One or more PEG molecules may be attached at different positions on the protein, and such attachment may be achieved by reaction with amines, thiols or other suitable reactive groups. The amine moiety may be, for example, a primary amine found at the N-terminus of a polypeptide or an amine group present in an amino acid, such as lysine or arginine. In some embodiments, the PEG moiety is attached at a position on the polypeptide selected from the group consisting of: a) the N-terminus; b) between the N-terminus and the most N-terminal beta strand or beta-like strand; c) a loop positioned on a face of the polypeptide opposite the target-binding site; d) between the C-terminus and the most C-terminal beta strand or beta-like strand; and e) at the C-terminus.

[000131] PEGylation may be achieved by site-directed PEGylation, wherein a suitable reactive group is introduced into the protein to create a site where PEGylation preferentially occurs. In some embodiments, the protein is modified to introduce a cysteine residue at a desired position, permitting site-directed PEGylation on the cysteine.
Mutations may be introduced into a protein coding sequence to generate cysteine residues. This might be achieved, for example, by mutating one or more amino acid residues to cysteine. Preferred amino acids for mutating to a cysteine residue include serine, threonine, alanine and other hydrophilic residues. Preferably, the residue to be mutated to cysteine is a surface-exposed residue. Algorithms are well-known in the art for predicting surface accessibility of residues based on primary sequence or a protein. Alternatively, surface residues may be predicted by comparing the amino acid sequences of binding polypeptides, given that the crystal structure of the framework, based on which binding polypeptides are designed and evolved, has been solved (see Himanen et al., Nature 2001;414:933-8) and thus the surface-exposed residues identified. PEGylation of cysteine residues may be carried out using, for example, PEG-maleimide, PEG-vinylsulfone, PEG-iodoacetamide, or PEG-orthopyridyl disulfide.

[000132] The PEG is typically activated with a suitable activating group appropriate for coupling to a desired site on the polypeptide. PEGylation methods are well-known in the art and further described in Zalipsky, S., et al., "Use of Functionalized Poly(Ethylene Glycols) for Modification of Polypeptides" in Polyethylene Glycol Chemistry:
Biotechnical and Biomedical Applications, J. M. Harris, Plenus Press, New York (1992), and in Zalipsky (1995) Advanced Drug Reviews 16: 157-182.

[000133] PEG may vary widely in molecular weight and may be branched or linear.
Typically, the weight-average molecular weight of PEG is from about 100 Daltons to about 150,000 Daltons. Exemplary weight-average molecular weights for PEG include about 1,000, Daltons, about 2, 000 Daltons, about 5,000 Daltons, about 10,000, Daltons, about 20,000 Daltons, about 40,000 Daltons, about 60,000 Daltons and about 80,000 Daltons.
In certain embodiments, the molecular weight of PEG is about 5,000 Daltons. Branched versions of PEG having a total molecular weight of any of the foregoing can also be used.
In some embodiments, the PEG has two branches. In other embodiments, the PEG has four branches.
In one embodiment, the PEG is a bis-PEG (NOF Corporation, DE-200MA).

[000134] Similar to antibodies, selective PEGylation of adnectins can be used to fine-tune (increase in increments) the half-life of the adnectins if necessary.

[000135] Conventional separation and purification techniques known in the art can be used to purify PEGylated FBS proteins, such as size exclusion (e.g., gel filtration) and ion exchange chromatography. Products may also be separated using SDS-PAGE. Products that may be separated include mono-, di-, tri-, poly- and un-PEGylated Adnectins, as well as free PEG.
The percentage of mono-PEG conjugates can be controlled by pooling broader fractions around the elution peak to increase the percentage of mono-PEG in the composition. About 90% mono-PEG conjugates represent a good balance of yield and activity.
IV. Targets

[000136] Exemplary in vivo target molecules which bind the 18F-labeled probes described herein are those associated with various diseases or conditions, such as a malignant disease, a cardiovascular disease, an infectious disease, an inflammatory disease, an autoimmune disease, or a neurological disease.

[000137] Provided herein are 18F labeled imaging agents, e.g., [18F]-moiety-FPPEGA, wherein the moiety binds specifically to a target molecule, such as a target protein on the surface of human cells. In certain embodiments, the moiety is a peptide; an antibody, or antigen binding portion thereof or a variant of an antibody; an alternative scaffold, such as an Fn3 (e.g., a human Fn3) domain, such as an FBS, e.g., a human 10Fn3 domain.
In certain embodiments, the moiety binds to a cell surface molecule, e.g., a cell surface molecule on a tumor cell or a cell in the tumor, e.g., a tumor infiltrating lymphocyte that is located in the tumor. In certain embodiments, the moiety binds to a cell surface molecule on an immune cell, e.g., a T cell (e.g., a Treg cell), a Teff cell, a B cell, a macrophage, a dendritic cell, an NK cell or a Langerhans cell.

[000138] In certain embodiments, an 18F labeled imaging agent comprises a moiety that binds specifically to an immuno-oncology target (receptor or ligand), such as a co-stimulatory receptor on an immune cell (e.g., T cell or NK cell) or an inhibitor on an immune cell (e.g., a T cell or NK cell), which targets modulate immune responses. In one embodiment, the moiety binds to one of the following molecules or ligand or receptor thereof:
an immunoglobulin super family (IgSF) member; a member of the B7 family, which includes B7-1, B7-2, B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6; a member of the TNF receptor superfamily or its ligand, e.g., CD40, CD40L, OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137, GITR, TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR/Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACT, APRIL, BCMA, LT13R, LIGHT, DcR3, HVEM, VEGI/TL1A, TRAMP/DR3, EDAR, EDA1, XEDAR, EDA2, TNFR1, Lymphotoxin a/TNF13, TNFR2, TNFa, LT13R, Lymphotoxin a 1132, FAS, FASL, RELT, DR6, TROY, NGFR (see, e.g., Tansey (2009) Drug Discovery Today 00:1); a protein that inhibits an immune cell (e.g., immune checkpoint inhibitors), such as CTLA-4, PD-1, PD-L1, PD-L2, and LAG-3, TIM-3, Galectin 9, CEACAM-1, BTLA, CD69, Galectin-1, TIGIT, CD113, GPR56, VISTA, 2B4, CD48, GARP, CD73, PD1H, LAIR1, TIM-1 ,TIM-4, CD39; a protein that stimulates an immune response, such as B7-1, B7-2, CD28, (CD137), 4-1BBL, GITR, GITRL, ICOS, ICOS-L, 0X40, 0X40L, CD70, CD27, CD40, DR3 and CD28H; any of the following cell surface molecules: KIR, cytokine or interleukin receptors, IL-6, IL-10, TGF-B, VEGF, CSF-1R, CD25 and IDO.

[000139] In some embodiments, the targeting molecule binds to an antigen or receptor of a pathogen, including but not limited to fungi, viruses, parasites and bacteria.
Examples of pathogenic viruses detectable by methods described herein include HIV, hepatitis (A, B, or C), herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus), adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus and arboviral encephalitis virus, human immunodeficiency virus (HIV), herpes virus, cytomegalovirus, rabies virus, influenza virus, hepatitis B
virus, Sendai virus, feline leukemia virus, Reo virus, polio virus, human serum parvo-like virus, simian virus 40, respiratory syncytial virus, mouse mammary tumor virus, Varicella-Zoster virus, Dengue virus, rubella virus, measles virus, adenovirus, human T-cell leukemia viruses, Epstein-Barr virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus, wart virus, blue tongue virus. Examples of bacteria and fungi include, Streptococcus agalactiae, Legionella pneumophilia, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Hemophilis influenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, Mycobacterium tuberculosis and Chlostridium tetani.
Some examples of pathogenic bacteria causing infections detectable by methods described herein include chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and gonococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and Lyme disease bacteria.

[000140] Some examples of pathogenic fungi causing infections detectable by methods described herein include Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neofonnans, Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor, absidia, rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum.

[000141] Some examples of pathogenic parasites causing infections detectable by methods described herein include Entamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, Nippostrongylus brasiliensis.

V. Biophysical and Biochemical Characterization [0001421 Binding of the protein, targeting molecules described herein to a target molecule may be assessed in terms of equilibrium constants (e.g., dissociation, KD) and in terms of kinetic constants (e.g., on-rate constant, k05 and off-rate constant, koff). A
protein targeting molecule will generally bind to a target molecule with a KD of less than 500 nM., 100 nM, 10 nM, 1 nM, 500 pM, 200 pM, or 100 pM, although higher KD values may be tolerated where the .koff is sufficiently low or the kon, is sufficiently high.
[000143] Exemplary assays for determining the binding affinity of a protein targeting molecule include, but are not limited to, solution phase methods such as the kinetic exclusion assay (KinExA) (Blake et al., IBC 1996; 271:27677-85; Drake et al, Anal Biochem 2004;
328:35-43), surface plasmon resonance (SPR) with the Biacore system (Uppsala, Sweden) (Weiford et al., Opt. Quant. Elect 1991; 23:1.; Morton and Myszka, Methods in Enzymology 1998; 295:268) and homogeneous time resolved fluorescence (HTRF) assays (Newton et al., Biomol Screen 2008; 13:674-82; Patel et al., Assay Drug Dev Technol 2008; 6:55-68).
[000144] In certain embodiments, biomolecular interactions can be monitored in real time with the Biacore system, which uses SPR to detect changes in the resonance angle of light at the surface of a thin gold film on a glass support due to changes in the refractive index of the surface up to 300 nm away. Biacore analysis generates association rate constants, dissociation rate constants, equilibrium dissociation constants, and affinity constants. Binding affinity is obtained by assessing the association and dissociation rate constants using a Biacore surface plasmon resonance system (Biacore, Inc.). A biosensor chip is activated for covalent coupling of the target. The target is then diluted and injected over the chip to obtain a signal in response units of immobilized material. Since the signal in resonance units (RU) is proportional to the mass of immobilized material, this represents a range of immobilized target densities on the matrix. Association and dissociation data are fit simultaneously in a global analysis to solve the net rate expression for a 1:1 bimolecular interaction, yielding best fit values for Icon, koc.f and R. (maximal response at saturation).
Equilibrium dissociation constants for binding, KB's are calculated from SPR measurements as lcoff¨ /lc ...-on =
[000145] In some embodiments, the protein targeting molecules described herein exhibit a K0 in the SPR affinity assay of 500 nM or less, 400 nM or less, 300 nM or less, 200 nIVI or less, 150 niVI or less, 100 nM or less, 90 nM or less, 80 UM or less, 70 nM or less, 60 TIM or less, 50 nM or less, 40 nM or less, 30 niVI or less, 20 nM or less, 15 nM or less, 10 nM or less, nM or less, or 1 nM or less.

[000146] it should be understood that the assays described herein above are exemplary, and that any method known in the art for determining the binding affinity between proteins (e.g., fluorescence based-transfer (FRET), enzyme-linked immunosorbent assay, and competitive binding assays (e.g., radioimmunoassays)) can be used to assess the binding affinities of the protein targeting molecules described herein.
VI. Formulations [000147] Further provided are compositions, e.g., a pharmaceutical compositions, containing one or a combination of 18F-labeled targeting agents, described herein, formulated together with a pharmaceutically acceptable carrier. Such compositions may include one or a combination of (e.g., two or more different) agents described herein. For example, a pharmaceutical composition described herein can comprise a combination of 18F-labeled targeting agent and a drug.
[000148] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, 18F-labeled targeting agent may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.
[000149] The pharmaceutical compounds described herein may include one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S.M., et al. (1977) J.
Pharm. Sci. 66:1-19).
Examples of such salts include acid addition salts and base addition salts.
Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

[000150] A pharmaceutical composition described herein also may include a pharmaceutically acceptable anti-oxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
[000151] Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
[000152] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum mono stearate and gelatin.
[000153] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions described herein is contemplated. Supplementary active compounds can also be incorporated into the compositions.
[000154] Pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
[000155] Sterile injectable solutions can be prepared by incorporating the 18F-labeled targeting agent in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration.
Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
[000156] The amount of 18F-labeled targeting agent which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of 18F-labeled targeting agent which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a detectable effect. Generally, out of one hundred per cent, this amount will range from about 0.01 per cent to about ninety-nine percent of active ingredient, preferably from about 0.1 per cent to about 70 per cent, most preferably from about 1 per cent to about 30 per cent of active ingredient in combination with a pharmaceutically acceptable carrier.
VII. Administration and Imaging [000157] The 18F-labeled targeting agents described herein are useful in a variety of in vivo imaging applications (e.g., for tissue or whole body imaging). in certain embodiments, the 18F-labeled targeting agent can be used to image target-posithe cells or tissues, e.g., target expressing tumors. For example, the labeled 18F-labeled targeting agent is administered to a subject in an amount sufficient to uptake the 18F-labeled targeting agent into the tissue of interest, The subject is then imaged using an imaging system such as PET for an amount of time appropriate for the 18F radionuclide, The 18F-labeled targeting agent-bound to cells or tissues expressing the targeting agent are then detected by the imaging system.
[000158] Typically, for imaging purposes it is desirable to provide the recipient with a dosage of protein or peptide that is in the range of from about 1 mg to 200 mg as a single intravenous infusion, although a lower or higher dosage also may be administered as circumstances dictate. Typically, it is desirable to provide the recipient with a dosage that is in the range of from about 1 mg to 10 mg per square meter of body surface area of the protein or peptide for the typical adult, although a lower or higher dosage also may be administered as circumstances dictate. Examples of dosages proteins or peptides that may be administered to a human subject for imaging purposes are about 1 to 200 mg, about 1 to 70 mg, about 1 to 20 mg, and about 1 to 10 mg, although higher or lower doses may be used.
[000159] In certain embodiments, administration occurs in an amount of 18F-radiolabeled -protein of between about 0.005ug/kg of body weight to about 50 g/kg of body weight per day, usually between 0.02ug/kg of body weight to about 3 g/kg of body weight.
The mass associated with a PET tracer is in the form of the natural isotope, namely 19F
for the 18F PET
tracer. A particular analytical dosage for the instant composition includes from about 0.5ug to about 100ug of an 18F-radiolabeled protein. The dosage will usually be from about lug to about 50ug of an 18F-radiolabeled protein.
[000160] Dosage regimens are adjusted to provide the optimum detectable amount for obtaining a clear image of the tissue or cells which uptake the 18F-labeled targeting agent. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to which the 18F-labeled targeting agent is to be administered. The specification for the dosage unit forms described herein are dictated by and directly dependent on (a) the unique characteristics of the targeting portion of the 18F-labeled targeting agent; (b) the tissue or cells to be targeted; (c) the limitations inherent in the imaging technology used.
[000161] For administration of the 18F-labeled targeting agent, the dosage used will depend upon the disease type, targeting compound used, the age, physical condition, and gender of the subject, the degree of the disease, the site to be examined, and others.
In particular, sufficient care has to be taken about exposure doses to a subject. Preferably, a saturating dose of 18T is administered to the patient. For example, the amount of radioactivity of 18F-labeled targeting agent usually ranges from 3.7 megabecquerels to 3.7 gigabecquerels, and preferably from 18 megabecquerels to 740 megabecquerels. Alternatively, the dosage may be measured by millicuries,for example. In some embodiments, the amount of 18F imaging administered for imaging studies is 5 to 10 mCi. In other embodiments, an effective amount will be the amount of compound sufficient to produce emissions in the range of from about 1-5 mCi.
[000162] Actual dosage levels of the active ingredients in the pharmaceutical compositions described herein may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired uptake of the 18F-labeled targeting agent in the cells or tissues of a particular patient, composition, and mode of administration, without being toxic to the patient. It will be understood, however, that the total daily usage of the 18F-labeled targeting agent of the present disclosure will be decided by the attending physician or other attending professional within the scope of sound medical judgment. The specific effective dose level for any particular subject will depend upon a variety of factors, including for example, the activity of the specific composition employed; the specific composition employed; the age, body weight, general health, sex, and diet of the host; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. In certain embodiments, the amount of 18F-radiolabeled probe administered into a human subject required for imaging will be determined by the prescribing physician with the dosage generally varying according to the quantity of emission from the 18F-radionuclide.
[000163] A composition described herein can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Preferred routes of administration for 18F-labeled targeting agent described herein include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. In certain embodiments, 18F-radiolabeled targeting compound is administered intravenously.
[000164] Alternatively, an 18F-labeled targeting agent described herein can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
[000165] In certain embodiments, the 18F-labeled targeting agent described herein can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. Agents may cross the BBB by formulating them, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S.
Patents 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V.V. Ranade (1989) J. Clin. Pharmacol. 29:685).
Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Patent 5,416,016 to Low et al.);
mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038);
antibodies (P.G. Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al. (1995) Am.
J. Physiol.
1233:134); p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K.
Keinanen; M.L.
Laukkanen (1994) FEBS Lett. 346:123; J.J. Killion; I.J. Fidler (1994).
[000166] The following illustrative procedure may be utilized when performing PET imaging studies on patients in the clinic. The patient is premedicated with unlabeled protein some time prior to the day of the experiment and is fasted for at least 12 hours allowing water intake ad libitum. A 20 G two-inch venous catheter is inserted into the contralateral ulnar vein for radiotracer administration concentration in the blood.
[000167] The patient is positioned in the PET camera and a tracer dose of the PET tracer of 18F-radiolabeled protein-based probe such as [18F] Example 9 compound (<20 mCi) is administered via i.v. catheter. Either arterial or venous blood samples are taken at appropriate time intervals throughout the PET scan in order to analyze and quantitate the fraction of unmetabolized PET tracer of [18F] Example 2 compound in plasma.
Images are acquired for up to 120 min. Within ten minutes of the injection of radiotracer and at the end of the imaging session, 1 ml blood samples are obtained for determining the plasma concentration of any unlabeled protein which may have been administered before the PET
tracer.

[000168] Tomographic images are obtained through image reconstruction. For determining the distribution of radiotracer, regions of interest (ROIs) are drawn on the reconstructed image including, but not limited to, the lungs, liver, heart, kidney, skin or other organs and tissue. Radiotracer uptakes over time in these regions are used to generate time activity curves (TAC) obtained in the absence of any intervention or in the presence of the unlabeled protein at the various dosing paradigms examined. Data are expressed as radioactivity per unit time per unit volume ( Ci/cc/mCi injected dose). TAC data are processed with various methods well-known in the field to yield quantitative parameters, such as Binding Potential (BP) or Volume of Distribution (VT), that are proportional to the density of unoccupied target positive tissue.
VIII. Kits and Articles of Manufacture [000169] Also provided are kits for producing the 18F-radiolabeled targeting compositions described herein and instructions for use. Kits typically include a packaged combination of reagents in predetermined amounts with instructions and a label indicating the intended use of the contents of the kit. The term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.
[000170] For example, in some embodiments, the kit contains the reagents necessary for the prosthetic group in condition to be fluorinated on site with 18F, and then linking the radiolabeled prosthetic group to the BFC-linked targeting molecule (e.g., protein or peptide) prior to administration.
[000171] In certain embodiments, a kit comprises one or more reagents necessary for forming an 18F labeled anti-PD-Li Adnectin in vivo imaging agent, such as a PD-Li Adnectin-PEG4-DBC0-18F, as further described herein. For example, a kit may comprise a first vial comprising anti-PD-Li Adnectin-PEG-4-DBCO and a second vial comprising [18F]FPPEGA. A kit may comprise a first vial comprising anti-PD-Li Adnectin-DBCO, a second vial comprising 4-PEG-tosyl-azide and a third vial comprising 18F in 018 water. The kits may further comprise vials, solutions and optionally additional reagents necessary for the manufacture of PD-Li Adnectin-PEG4-DBC0-18F.
[000172] In some embodiments, the kit can further contain at least one additional reagent (e.g., pharmaceutically acceptable carrier). In some embodiments, the kit includes the reaction precursors to be used to generate the labeled probe according to the methods disclosed herein. The components of the kit can be tailored to the particular biological condition to be monitored as described herein. The kit can further include appropriate buffers and reagents known in the art for administering various combinations of the components listed above to the host cell or host organism. The imaging agent and carrier may be provided in solution or in lyophilized form. When the imaging agent and carrier of the kit are in lyophilized form, the kit may optionally contain a sterile and physiologically acceptable reconstitution medium such as water, saline, buffered saline, and the like.
Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
IX. Uses [000173] Methods of imaging using 18F-labeled targeting agents are provided herein.
Positron emission tomography (PET) tracers such as the present 18F-radiolabeled protein-based PET probes can be used with currently available PET technology for use in exploratory and diagnostic imaging applications in vitro and in vivo. Imaging techniques and equipment for 18F imaging by PET scanning are well known in the art (see, e.g., U.S.
Pat. Nos.
6,358,489; 6,953,567; Page et al., Nuclear Medicine And Biology, 21:911-919, 1994; Choi et al., Cancer Research 55:5323-5329, 1995; Zalutsky et al., J. Nuclear Med., 33:575-582, 1992) and any such known PET imaging technique or apparatus may be utilized.
[000174] In vivo applications of the imaging methods provided herein include disease diagnosis, monitoring of disease progression, prognosis, determining likelihood of a subject to respond to a treatment, determining eligibility to a treatment, monitoring of clinical response to therapy, clinical evaluation and dose selection of therapeutic compounds, preclinical studies of potential drug candidates in animal models, and the study of regional distribution and concentration of target molecules in tissues and organs. In vitro applications include screening of drug candidates in cell assays (e.g., competition assays, affinity assays, etc.) [000175] In some embodiments, the 18F-labeled targeting agents can be used to determine the relationship between level of tissue occupancy by candidate therapeutic compounds and clinical efficacy in patients; to determine dose selection for clinical trials of drug candidates prior to initiation of long term clinical studies; and to compare potencies of different drug candidates.
[000176] In some embodiments, the 18F-radiolabeled targeting compound is used in a method for in in vivo imaging normal or diseased tissues and/or organs (e.g., lungs, heart, kidneys, liver, and skin). For example, the 18F-radiolabeled targeting compound is administered to a subject in an amount effective to result in uptake of the 18F-radiolabeled targeting compound into the cells or tissue of interest. The subject is then introduced to an appropriate imaging system (e.g., PET system) for a sufficient amount of time to allow detection of the 18F-radiolabeled targeting compound. The location of the detected signal from the radiolabeled targeting compound can be correlated with the location of the cells or tissue of interest. In some embodiments, the dimensions of the location can be determined as well. In vivo imaging is described herein. See also U.S. Pat. Nos. 6,126,916;
6,077,499; 6,010,680;
5,776,095; 5,776,094; 5,776,093; 5,772,981; 5,753,206; 5,746,996; 5,697,902;
5,328,679;
5,128,119; 5,101,827; and 4,735,210, each incorporated herein by reference.
[000177] Accordingly, in certain aspects, provided is a method of obtaining an image of an 18F-radiolabeled protein-based probe, the method comprising administering the radiolabeled protein-based probe to a subject, and imaging in vivo the distribution of the 18F-radiolabeled protein-based probe by PET.
[0001781 In certain embodiments, the subject is a mammal, for example, a human, dog, cat, ape, monkey, rat, or mouse.
[000179] In certain aspects, provided is a method of diagnosing the presence of a disease in a subject, the method comprising administering to a subject in need thereof an 18F-radiolabeled protein-based probe which binds to a target molecule associated with the presence of the disease, and obtaining a radio-image of at least a portion of the subject to detect the presence or absence of the 18F-radiolabeled protein-based probe.
[000180] In some embodiments, the disease is a solid cancer, hematopoietic cancer, hematological cancer, autoimmune disease, neurodegenerative disease, cardiovascular disease or pathogenic infection.
[000181] PET imaging with an 8F-radiolabeled targeting compound may be used to qualitatively or quantitatively detect the targeting compound. An 8F-radiolabeled targeting compound imaging agent may be used as a biomarker, and the presence or absence of a positive signal in a subject may be indicative that, e.g., the subject would be responsive to a given therapy, e.g., a cancer therapy, or that the subject is responding or not to a therapy.
[000182] In some embodiments, the steps of this method can be repeated at determined intervals so that the location and/or size of the disease can be monitored as a function of time and/or treatment. In certain embodiments, the 18F-radiolabeled targeting compound can be used in a subject undergoing treatment (e.g., chemotherapy, etc.), to aid in visualizing response to the treatment. For example, the 18F-radiolabeled targeting compound is typically visualized and sized prior to treatment, and periodically (e.g., daily, weekly, monthly, intervals in between these, and the like) during treatment to monitor the progression or regression of the disease in the patient.
[000183] Accordingly, in certain aspects, provided is a method of monitoring the progress of a disease in a subject in need thereof, the method comprising administering to the subject an 18F-radiolabeled protein-based probe which binds to a target molecule associated with the presence of the disease at a first time point and obtaining an image of at least a portion of the subject to determine the amount of diseased cells or tissue, and administering to the subject the 18F-radiolabeled protein-based probe at one or more subsequent time points and obtaining an image of at least a portion of the subject at each subsequent time point (e.g., same portion as the first time point).
[000184] In certain embodiments, the size of a tumor can be monitored in a subject undergoing cancer therapy (e.g., chemotherapy, radiotherapy) and the extent of regression of the tumor can be monitored in real-time based on detection of 18F-radiolabeled tumor targeting.
[000185] In some embodiments, the methods herein are used to evaluate the patient's response to therapy. In some embodiments, the methods are used to select or modify the dosage of therapeutic compounds. In some embodiments, the methods are used to monitor the uptake of the 18F-radiolabeled targeting compound in normal tissues to analyze toxicity or patient to patient variation. In some embodiments, the methods are used to monitor drug efficacy or to detect drug resistance.
[000186] In some embodiments, the radiolabeled compounds are administered to mammals, preferably humans, in a pharmaceutical composition, either alone or in combination with pharmaceutically acceptable carriers or diluents according to standard pharmaceutical practice. Such compositions can be administered orally or parenterally, including the intravenous, intramuscular, intraperitoneal, subcutaneous, rectal and topical routes of administration. In certain embodiments, administration is intravenous. In certain embodiments the radiolabeled compound is administered via intravenous injection within less than one hour of synthesis.
[000187] In some embodiments, the biological activity of the 18F-radiolabeled targeting agent in vivo may be measured in terms of organ-specific uptake by biodistribution studies and dynamic small animal PET imaging studies in an appropriate animal model. For example, for biodistribution studies, a group of animals are injected with the 18F-radiolabeled targeting agent and the subsets of the animals are sacrificed at one or more time intervals (e.g., 5 min., min., 30 min., 60 min., 2 h). Organs and tissues of interest are rapidly excised and weighed, and radioactivity determined. Accumulated radioactivity in organs and selected tissues is calculated as the percentage of injected dose (%ID).
[000188] In some embodiments, the 18F-radiolabeled targeting agent provided herein is used in vitro as a screening tool to select compounds for use in treating tissues or cells. For example, in some embodiments, diseased cells are incubated with the 18F-radiolabeled targeting compound during or after exposure to one or more candidate drugs.
The ability of the drug candidate to affect the disease can be imaged over time using the 18F-radiolabeled targeting compound.
[000189] For example, the integrity of biological activity of the 18F-radiolabeled targeting agent in vitro in terms of specific binding to the selected target molecule and uptake of the radiolabeled composition is assessed in a cell line expressing the target molecule. For binding and cell association assays, cells are incubated at 4 C or 37 C for an appropriate time with the 18F-radiolabeled targeting composition. Nonspecific binding is determined by the addition of an excess of unlabeled targeting agent. The extent of specific binding is calculated by subtracting the nonspecific binding from the total binding.
Uptake is expressed as a percentage of the total added dose of targeting agent to the cells per microgram of protein (%ID/[tg cell protein) [000190] In a related aspect, the present invention provides a diagnostic or radiopharmaceutical composition for in vivo or in vitro, which includes an 18F-radiolabeled protein-based probe, and a pharmaceutically acceptable carrier.

Incorporation by Reference [000191] All documents and references, including patent documents and websites, described herein are individually incorporated by reference to into this document to the same extent as if there were written in this document in full or in part.
[000192] The invention is now described by reference to the following examples, which are illustrative only, and are not intended to limit the present invention. While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one of skill in the art that various changes and modifications can be made thereto without departing from the spirit and scope thereof.

Preparation of 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate ri 0-r ri 0, 0 \ S/, 0 \ 0 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate 0--g .
N
j-0 /--/

/--/ 0-r /--/
0 Na 0.... / µN=N+:1\1- /-0 SCD _No,. 0, ,0¨/
= Et0H, reflux \S, 0 \O
2-(2-(2-(2-azidoethoxy)ethoxy)ethoWethyl methylbenzenesulfonate [000193] A mixture of ((oxybis(ethane-2,1-diy1))bis(oxy))bis(ethane-2,1-diy1) bis(4-methylbenzenesulfonate) (5 g, 9.95 mmol) and SODIUM AZIDE (0.647 g, 9.95 mmol) were dissolved in ethanol (50 mL) and the reaction was refluxed at 90 C over a 17 hour period.
The solvent was removed using partial vacuum and then loaded onto a 40 gram silica cartridge and was purified using flash chromatography (IscoCombiFlash - eluted using a linear gradient method starting from 10% ethyl acetate in hexanes going to a 90% ethyl acetate in hexanes over a 45 minute period. The pooled fractions were checked by TLC and combined to give 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate as a colorless oil. Due to the reactive nature of the 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate product this material was used "as is" without any further characterizations.

Preparation of 3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine /--/
0-r /--/
F o_r 3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine rj N3 rj HO
0¨/-0 /--/
0¨/ F N
F 0¨r . NaH, DMF NC--\ /
3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine [000194] To the suspension of sodium hydride (0.129 g, 3.21 mmol) in DMF (10 mL) at 0 C was dropwise added a stirring solution of 2-fluoropyridin-3-ol (0.363 g, 3.21 mmol) in DMF (5 mL), then followed by the dropwise addition of the solution of 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (1.00 g, 2.68 mmol) in DMF (5 mL). The suspension was held at 0 C for 10 min, then brought to ambient temperature for 1 hour, followed by addition heating at 60 C for 4 hours. Solvent was removed in vacuo. 100 ml of ethyl acetate was added followed by 3 separate wash extractions with concentrated brine solution. The organic layer was dried over sodium sulfate, filtered and concentrated.

The crude material was purified using flash chromatography (IscoCombiFlash -eluted with - 50% Et0Ac in Hex) to give a colorless oil. 3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine (702 mg, 2.233 mmol, 83 %
yield) was isolated as a clear oil. 1-F1 NMR (400MHz, CHLOROFORM-d) 6 7.75 (dt, J=4.9, 1.6 Hz, 1H), 7.33 (ddd, J=10.0, 8.1, 1.5 Hz, 1H), 7.10 (ddd, J=7.9, 4.9, 0.7 Hz, 1H), 4.30 - 4.16 (m, 2H), 3.95 - 3.83 (m, 2H), 3.80 - 3.61 (m, 10H), 3.38 (t, J=5.1 Hz, 2H) 13C NMR
(101MHz, CHLOROFORM-d) d 142.3, 137.7, 137.5, 123.4, 123.4, 121.7, 121.6, 77.3, 76.7, 70.9, 70.7, 70.6, 70.0, 69.4, 69.0, 50.6 19F NMR (400MHz, CHLOROFORM-d) 6 -83.55. HRMS
(ESI) Theory:C13H2OFN404+ m/z 315.464; found 315.1463 Preparation of 3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-nitropyridine /
o¨r /--/
02N o_r N\ -----\ /
3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-nitropyridine / /
0-r /--/ HO
1 0¨/-0 0¨r 0NN

. / 02N o_r Sc).
= NaH, DMF
\ /
3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-nitropyridine [000195] Sodium hydride (0.121 g, 3.01 mmol) (60% suspension in oil) was dissolved in DMF (7.0 mL) and the resulting suspension was cooled to 0 C. A solution of 2-nitropyridin-3-ol (0.384 g, 2.74 mmol) in DMF (1.5 mL) was added slowly, followed by the drop wise addition of 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (1.023 g, 2.74 mmol) in DMF (1.5 mL). The suspension was held at 0 C for 10 minutes, then brought to ambient temperature for 2 hours followed by heating 60 C for a 72 hour period. Reaction was quenched with 10 ml of DI water, followed by ethyl acetate extraction (3 x 10 mL).
Pooled Et0Ac extracts were washed with a concentrated brine solution (10 mL), dried over sodium sulfate, filtered and evaporated under reduced pressure to give a light yellow oil. The crude was purified by flash chromatography. 24 g silica cartridge, 25 mL/min, starting from 10% ethyl acetate in hexanes, followed by a linear change to 50% ethyl acetate in hexanes over a 25 minute period. After this time the gradient was held at this solvent composition for minutes then changed to 100% ethyl acetate over a 10 minute period. 3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-nitropyridine was eluted between 30-40 minute portion of the chromatogram and the pooled fractions were evaporated under reduced pressure, then under vacuum for 2 hours to give 3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-nitropyridine (687 mg, 1.973 mmol, 72.0 % yield) as a light yellow oil. 1-FINMR (400MHz, CHLOROFORM-d) 6 8.11 (dt, J=4.9, 1.6 Hz, 1H), 7.60 (ddd, J=10.0, 8.1, 1.5 Hz, 1H), 7.52 (ddd, J=7.9, 4.9, 0.7 Hz, 1H), 4.30 - 4.16 (m, 2H), 3.95 - 3.83 (m, 2H), 3.80 -3.61 (m, 10H), 3.38 (t, J=5.1 Hz, 2H) 13C NMR (101MHz, CHLOROFORM-d) d 147.3, 139.5, 128.4, 124.4. 71.1, 70.7, 70.6,70.0, 69.9, 69.3, 50.7. HRMS (ESI) Theory:C13H20N506+
m/z 342.1408; found 342.1409 Synthesis of 3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-bromopyridine /--/
0-r /--/
Br 0¨r N
\/
3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-bromopyridine 0-r /--/ HO
1 0¨/-0 0¨r BrN
Br 0¨/ /¨

' .
'0 ilfr NaH, DMF NJ\ ---.--\ /
[000196] To the suspension of sodium hydride (NaH, 25.7 mg, 0.643 mmol) in dimethylformamide (DMF, 5 mL) at 0 C was dropwise added a solution of 2-bromopyridin-3-ol (112 mg, 0.643 mmol) in DMF (1 mL), followed by the dropwise addition of the solution of 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (200 mg, 0.536 mmol) in DMF (1 mL). The suspension was held at 0 C for 10 minutes, then brought to ambient temperature and held for 1 hour, followed by heating to 60 C for 4 hours. Upon completion of heating, the solvent of the crude reaction mixture was removed in vacuo. The crude reaction was reconstituted in 50 mL of ethyl acetate , washed with 2 x 50 mL of a aqueous brine solution and the organic layer was dried over magnesium sulfate, filtered and concentrated in vacuo. The crude reaction was purified using reverse-phase HPLC to give3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-bromopyridine, TFA (112 mg, 0.229 mmol, 42.7 % yield) as a light yellow oil. HRMS ESI m/z (M+H), Theory C13H20BrN404 375.0664 found 375.0662; 1-FINMR (400MHz, DMSO-d6) 6 7.97 (dd, J=4.6, 1.5 Hz, 1H), 7.54 (dd, J=8.2, 1.6 Hz, 1H), 7.40 (dd, J=8.1, 4.6 Hz, 1H), 4.24 (dd, J=5.3, 3.9 Hz,2H), 3.85 -3.78 (m, 2H), 3.68 - 3.62 (m, 2H), 3.62 - 3.52 (m, 8H), 3.42 - 3.34 (m, 2H).

Scheme for synthesis of trimethylanilium compound ri N3 ri 0¨r /--/ HO
0¨/-0 _/-0 I

OS
. /C) F N
F
' . _30,.. 0¨/-0 /
'0 ilfr NaH, DMF N5 H
N(40% aqueous) V K2CO3, DMSO

N 1 (3 OTf 8 /------.3 N3 N L/C)----/---C) 6, 3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-N,N,N-trimethylpyridin-2-aminium Synthesis of 3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-N,N-dimethylpyridin-2-amine Nr 1----/

No/
I Z
3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoWethoW-N,N-dimethylpyridin-2-amine o¨r o¨r /--/ H
N(40% aqueous) ---N/ /-0/¨/
___________________________________ Diw N5 K2003, DMSO N \ ---/
3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-N,N-dimethylpyridin-2-amine [000197] A mixture of 3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine (160 mg, 0.509 mmol), potassium carbonate (K2CO3, 84 mg, 0.611 mmol) and dimethylamine (40% in water, 0.097 mL, 0.764 mmol) in dimethylsulfoxide (DMSO, 2.5 mL) were heated in a sealed pressure-proof vessel at 110 C for 14 hours. Upon completion of heating, the solvent of the crude reaction mixture was removed in vacuo.
The crude reaction was reconstituted in 50 mL of ethyl acetate, washed with 2 x 50 mL of a aqueous brine solution and the organic layer was dried over magnesium sulfate, filtered and concentrated in vacuo. The crude reaction was purified using normal-phase chromatography to give 3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-N,N-dimethylpyridin-2-amine (140 mg, 0.413 mmol, 81 % yield) as a colorless oil. 1H NMR (400MHz, CHLOROFORM-d) 7.86 (dd, J=4.9, 1.5 Hz, 1H), 7.02 (dd, J=7.8, 1.5 Hz, 1H), 6.73 (dd, J=7.8, 4.9 Hz, 1H), 4.20 - 4.07 (m, 2H), 3.98 - 3.86 (m, 2H), 3.81 - 3.61 (m, 9H), 3.38 (t, J=5.1 Hz, 2H), 3.13 - 2.94 (m, 6H), 1.69 (s, 2H). HRMS (ESI) Theory:C15H26N504+ m/z 340.1980; found 340.1979.

Synthesis of 3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-N,N,N-trimethylpyridin-2-aminium NI GOTf N----N xe 0 07`-..7C)0,--=,N3 /
----6, 3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-N,N,N-trimethylpyridin-2-aminium F 0µµc (-1 µ sµ-, /........./ N3 , b \ N
,0---/--0 r F N i e0Tf ________________________________________ v.
N 0.,_7(:)N3 C)---/----C) 0 16-- --,7'07 Toluene I
3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-N,N,N-trimethylpyridin-2-aminium [000198] Methyl trifluoromethanesufonate (0.065 mL, 0.589 mmol) was added to the solution of 3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-N,N-dimethylpyridin-2-amine (40 mg, 0.118 mmol) in toluene (1.5 mL) in a sealed container under a steady stream of nitrogen. The reaction mixture was stirred at room temperature over a 14 hour period. The solvent was removed and the resultant residue was washed with 2x 10 ml of ether, azeotropically dried with 2 x 1 ml of dichloromethane and dried under high-pressure vacuum overnight to give 3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-N,N,N-trimethylpyridin-2-aminium, trifluoromethanesulfonate salt in quantitative yield as a thick colorless oil.
LCMS m/z 354.33; 1-H NMR (400MHz, DMSO-d6) 6 8.24 - 8.17 (m, 1H), 7.98 (d, J=8.3 Hz, 1H), 7.75 (ddd, J=8.2, 4.6, 3.2 Hz, 1H), 4.44 (br. s., 2H), 3.88 (d, J=3.9 Hz, 2H), 3.69 - 3.45 (m, 21H).

The synthesis of the [18F]-3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine using 3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-N,N,N-trimethylpyridin-2-aminium, trifluoromethanesulfonate salt N,-,0,-,C) c) X r(D
X= NO2, Br or N(Me)3 }.) Ni"---K.2.2.2 [1-. F]KF , 1D2MOS C
F.2%..._,3 mins iq 0 18F ((:) IZ) Synthesis of [18F] 3 (2 (2 (2 (2 azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine [000199] An aqueous [18F]-Fluoride solution (2.0 m1,33.3 GBq/ 900 mCi) was purchased from P.E.T. Net Pharmaceuticals in West Point PA and directly transferred to a Sep-Pak light QMA [The Sep-Pak light QMA cartridge was pre-conditioned sequentially with 5m1 of 0.5 M potassium bicarbonate, 5 ml of deionized water, and 5 ml of MeCN before use.] Upon completion of this transfer, the aqueous [18F] fluoride was released from the QMA Sep-Pak by the sequential addition of potassium carbonate ( 15 mg/ml; 0.1m1) followed by a mixture of potassium carbonate ( 30 mg/ml, 0.1 ml), 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane ( 15 mg, 0.04 mmol) and 1.2 ml of MeCN. The solvent was evaporated under a gentle stream of nitrogen at 90 C and vacuum. Azeotropic drying was repeated twice with lml portions of acetonitrile to generate the anhydrous K.2.2.2/K[18F]F
complex. 3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-N,N,N-trimethylpyridin-2-aminium, trifluoromethanesulfonate salt (2 mg, 5.6 [tmol) was dissolved in 500 microliters of DMSO and added to the dried cryptand. This solution was heated at 120 C for 10 minutes.
After this time the crude reaction mixture was diluted with 3 ml of DI water.
The entire contents of the crude reaction mixture was then transferred, loaded and purified using reverse phase HPLC and the following conditions: HPLC Column: Luna C18 250 x 10 Solvent A:
0.1 % TFA in DI water; solvent B: 0.1 % TFA in acetonitrile at a flow rate of 4.6 ml/minute using isocratic method 32% B while the UV was monitored at 280 nm.
azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine was isolated at the 24 min mark of the chromatogram. and was collect over a 2 minute period. This product was collected into a 100 ml flask that contained 10 ml of DI water and the entire contents were delivered to a Sep-Pak Vac tC18 6 cc lg sep pack from Waters. 6.1 GBq/164 mCi of [18F]-3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine was isolated from this reaction. This was released from the sep-pak using 3 ml of ethanol and this solution was reduced with 98 C
heat source, a gentle stream of nitrogen, and vacuum over a 15 minute period until only a film remained in this vial. The final product was reconstituted in100% lx PBS
buffer and is stable in this media for over 1 hour at 37 C.
[000200] The [18F]-3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine may be used to generate 18F labeled biologic products by taking advantage of the "click" azide-alkyne reaction with the appropriate biologic containing an alkynes.

Production of 18F-radiolabeled protein using "Click Chemistry"
[000201] In this example, [18F]-3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine was used to radiolabel a target protein as illustrated in Figure 1 and 5.
A. Fluorination of the 4-PEG-tosyl-azide Precursor to Form 1-18F1-FPPEGA
[000202] 900 mCi of 18F in 180 water (3m1) activity (purchased from IBA
Molecular) was transferred directly into a micro vial ( no QMA) that contained 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane (2.8 mg, 7.44 [tmol) and potassium carbonate (1.7 mg, 0.012 mmol). An additional 2.0 ml of acetonitrile was transferred into this crude reaction mixture and the entire mixture was azeotropically dried. This was completed by evaporating the solution using a 98 C oil bath, and applying a gentle stream of N2 and partial vacuum.
The solution's volume was reduced to about 2 ml. An additional 2 ml of acetonitrile was added and the process was repeated 3 times over a 40 minute period. When the volume of the liquid was reduced to less than 0.3 ml, a 0.7 ml aliquot of acetonitrile was added and the solution reduced by further azeotropic distillation until the volume was ¨0.1 ml. An additional 0.9 ml of acetonitrile was added and this process was completed until a white solid was formed. This process took ¨55 minutes. During the final procedure, the vial was removed from the oil bath before the solution had gone to dryness and the residue in the vial was placed under full vacuum (no N2 flow) at room temperature for 20 minutes.
Total time for transfer and drying of [18F]-FPPEGA cryptand mixture was 65 min.
[000203] To the dried [18F]-FPPEGA cryptand mixture was added 3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-nitropyridine (2 mg, 5.86 [tmol) dissolved in 500 microliters of DMSO and this mixture was heated at 120 C for 10 minutes.
After this time the crude reaction mixture was diluted with 3 ml of DI water and the entire contents were then transferred and loaded onto the following HPLC column and conditions:
HPLC
Column: Luna C18 250 x 10 mm; Solvent A: 0.1 % TFA in DI water; Solvent B: 0.1 % TFA
in acetonitrile; flow rate 4.6 ml/min; pressure 1820 PSI; isocratic method 32%
B; UV - 280 nm. The [18F]-3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine ([18F]-FPPEGA) product was isolated at the 24 minute mark of the chromatogram and was collect over a 2 minute period. This product was collected into a 100 ml flask that contained 15 ml of DI water and the entire contents were delivered to a Sep PakVac tC18 6 cc 1 g sep pack.
PN WAT036795. The [18F]-FPPEGA was released from the Sep Pak using 2.5 ml of ethanol and this solution was reduced with 98 C N2 and vacuum over a 15 minute period until dryness. This compound was dissolved in 0.1 ml 1 X PBS (phosphate buffered saline). This product was analyzed using a Varian HPLC HPLC Column Luna C18 (2) 4.6 x 150 mm Solvent A: 0.1 % TFA in DI water; Solvent B: 0.1 % TFA in acetonitrile; flow rate 1.0 ml/min; gradient method 0 min 90% A 10% B; 15 mins 30% A 70% B; 17 mins 30% A
70%
B; 18 mins 90% A 10% B; 20 mins 90% A 10% B; UV - 280 nm. 220 mCi of [18F]-FPPEGA
was isolated.
B. Preparation of E01-4PEG-DBCO
[000204] An FBS protein, E01 Adnectin with the following amino acid sequence;
GVSDVPRDLEVVAATPTSLLISWRAQLSPSFYYRITYGETGGNSPVQEFT
VPNDVMTATISGLKPGVDYTITVYAVTTHGVYFYSPISINYRTPC (E01; SEQ ID NO:
1) containing the C-terminal amino acids PC was used.
[000205] As maleimide chemistry was used to link the targeting protein to PEG4-DBCO, the E01 Adnectin was first modified by adding a proline followed by a cysteine at its C-terminus using routine recombinant techniques. A 4-fold molar excess of Maleimide-PEG4-DBCO
(Click Chemistry Tools) was dissolved in DMSO and added to the purified modified E01 Adnectin in the presence of 1mM TCEP. Final DMSO concentrations did not exceed 5% in the conjugation mixtures. The conjugation mixture was left at room temperature for one hour before mass spec analysis. After MS confirmation of conjugation, the sample was purified by size-exclusion chromatography using a HiLoad 26/60 Superdex 75 column (GE
Healthcare) equilibrated in PBS pH 7.2.
C. Coupling of I-18F1-FPPEGA to Adnectin [000206] 0.2 ml of a 5.4 mg/ml solution of the E01-4PEG-DBCO adnectin solution (prepared as described in Section B) was incubated with 200 mCi of 0.1 ml of the [1-8fl-FPPEGA
(Example 1) in 1 x PBS buffer. The solution was gently mixed by pipetting the crude reaction up and down several times and was incubated together for 45 minutes at 45 C or at room temperature. The contents of this crude reaction mixture were purified using a SEC column.
Superdex 200 0.5 ml/min 1 X PBS buffer and the [18F]E01-4PEG-DBCO-FPPEGA
product was isolated at the 37 min mark of the chromatogram over a 2 minute period.
[000207] [18F]E01-4PEG-DBCO-FPPEGA was analyzed via SEC with co-injection of non-radioactive standard, RP HPLC using a PLRPS column and gel electrophoresis.
[000208] Size Exclusion Chromatography (SEC) was performed with the following parameters:

Superdex 200 column; Solvent 100% 1X PBS buffer; 0.5 ml/min 280 UV;
Reverse phase HPLC
Column: PLRPS 8 micron 1000 A 4.6 x 250 mm Solvent A: 0.1% formic acid in DI water Solvent B: Acetonitrile Flow rate: 1 ml/min Pressure: 1351 PSI
Gradient:
0 min 90% A 10% B
30 min 45 %A 55% B
32 min 25% A 75% B
36 min 25% A 75% B
50 min 90% A 10% B
[000209] 15 mCi [18F]E01-4PEG-DBCO-FPPEGA was isolated with a radiochemical purity (RCP) of >99% via both SEC and RP HPLC calculations, and with a specific activity of 0.6 mCi/nmol, when the reaction was conducted at 45 C. When conducting the reaction at room temperature, 5.72 mCi was obtained. Specific activity of the [18F]-FPPEGA was 0.512 mCi/nmol and RCP of 85.7% 3 hours post the end of its synthesis, when conducting the reaction at 45 C or at room temperature, respectively. Specific activity was measured via Nanodrop (see http://www.nanodrop.com). The product co-eluted with non-radioactive standard on both SEC and PLRPS. Gel electrophoresis confirmed an 18F product consistent with an 11 kDa molecular weight standard.
[000210] The 18F-radiolabeled E01-4PEG-DBCO can be used in a variety of in vitro and/or in vivo imaging applications, including diagnostic imaging, basic research, and radiotherapeutic applications. Specific examples of possible diagnostic imaging and radiotherapeutic applications, include determining the location, the relative activity and/or quantifying of PD-Li positive tumors, radioimmunoassay of PD-Li positive tumors, and autoradiography to determine the distribution of PD-Li positive tumors in a mammal or an organ or tissue sample thereof.
[000211] In particular, the 18F-radiolabeled E01-4PEG-DBCO is useful for positron emission tomographic (PET) imaging of PD-Li positive tumors in the lung, heart, kidneys, liver and skin and other organs of humans and experimental animals. PET imaging using the 18F-radiolabeled E01-4PEG-DBCO can be used to obtain the following information:
relationship between level of tissue occupancy by candidate PD-Li tumor-treating medicaments and clinical efficacy in patients; dose selection for clinical trials of PD-Li tumor-treating medicaments prior to initiation of long term clinical studies; comparative potencies of structurally novel PD-Li tumor-treating medicaments; investigating the influence of PD-Li tumor-treating medicaments on in vivo transporter affinity and density during the treatment of clinical targets with PD-Li tumor-treating medicaments; changes in the density and distribution of PD-Li positive tumors during effective and ineffective treatment.
[000212] For example, inhibition of PD-Li can calculated based on the change of BP or VT
by equilibrium analysis in the presence of PD-Li tumor-treating medicament at the various dosing paradigms as compared to the BP or VT in the unmedicated state.
Inhibition curves are generated by plotting the above data vs. the dose (concentration) of PD-Li tumor-treating medicament. Inhibition of PD-Li positive tumors is then calculated based on the maximal reduction of PET radioligands VT or BP that can be achieved by a blocking drug at Emax, Tmax or Tm,õ and the change of its non-specific volume of distribution (VND) and the BP in the presence of PD-Li tumor-treating medicaments at the various dosing paradigms as compared to the BP or VT in the unmedicated state. The ID50 values are obtained by curve fitting the dose-rate/inhibition curves.

In vitro Differentiation of PD-Li-positive cells from PD-Li-negative cells with an anti-PD-Li Adnectin imaging agent [000213] In this experiment, the 18F-radiolabeled E01-4PEG-DBCO was tested for its ability to discriminate between PD-Li -positive cells and PD-Li-negative cells in vitro.
[000214] lx106 PD-Li-positive L2987 human lung carcinoma cells or PD-Li-negative HT-29 human colorectal adenocarcinoma cells were placed into 5mL culture tubes (n=3 tubes per condition). 18F-radiolabeled E01-4PEG-DBCO solution was prepared in PBS + 0.5%
BSA at a concentration of 300nCi/200 L. Portions of this solution were supplemented with either cold (unlabeled) E01 Adnectin or cold (unlabeled) Adnectin (control) to a final concentration of 450nM. Cell samples were centrifuged for 5min at 200xg and then resuspended in 2000_, of the appropriate 18F-radiolabeled E01-4PEG-DBCO solution and incubated on ice for 1 hour. After the incubation period, cell samples were centrifuged at 200xg and the supernatant was discarded. Cell pellets were resuspended in 1 mL PBS + 0.5%
BSA and the wash procedure repeated for a total of 3 washes. Following the final wash, cells were again centrifuged at 200xg and the supernatant was discarded. The radioactivity of the remaining cell pellets was then measured by gamma counter.
[000215] The results indicated that ell labeling was specific, as evidenced by differential association of 18F-radiolabeled E01-4PEG-DBCO with PD-Li-positive L2987 cells compared to PD-Li-negative HT-29 cells (cell associated radioactivity was 44.6x higher in PD-L1-positive L2987 cells). Specificity was further confirmed as evidenced by a marked reduction in cell-associated 18F-radiolabeled E01-4PEG-DBCO when co-incubated with excess 450nM
cold (unlabeled) E01 Adnectin (99.6% reduction). Cell associated 18F-E01 was minimally reduced (9.9% reduction, not significant) when cells were co-incubated with 450nM cold (unlabeled) non-PD-Li binding adnectin.
[000216] Taken together, these results demonstrate the ability of the 18F-radiolabeled E01-4PEG-DBCO to differentiate PD-L1(+) vs. PD-L1(-) cells in vitro.

Distinguishing PD-Li-positive tumors from PD-Li-negative tumors in vivo with an anti-PD-Li Adnectin imaging agent [000217] In this experiment, the 18F-radiolabeled PD-Li Adnectin with the following amino acid sequence:
EVVAATPTSLLISWSYDGPIDRYYRITYGETGGNSPVQEFTVPPDQKTATISGLKPGV
DYTITVYAVRLEEAHYNREFPISINYRTPC (A02; SEQ ID NO:2) was produced as described in Example 9, was tested for the ability to discriminate between PD-Li-positive tumors and PD-Li-negative tumors in mice.
[000218] Mice bearing bilateral xenograft tumors were produced by introducing lx106 PD-L1(+) L2987 human lung carcinoma cells and 1.5x106 PD-L1(-) HT-29 human colon carcinoma cells subcutaneously on opposite sides of the mouse. Once tumors reached approximately 300mm3 (approximately 2-3 weeks after cell implantation) animals were selected for imaging. For imaging, animals were placed under anesthesia with 2% isoflurane and tail vein catheters were installed. Mice were then placed into a custom animal holder with capacity for 4 animals, where they remained under anesthesia for the duration of the study. The animal holder was transferred to the microPET F120TM scanner (Siemens Preclinical Solutions, Knoxville, TN). The axial field of view of this instrument is 7.6 cm.
With this limitation, animals were positioned such that the scanning region was from immediately in front of the eyes to approximately the base of the tail.
[000219] A 10-minute transmission image was first acquired using a 57Co point source for the purpose of attenuation correction of the final PET images. Following the transmission scan, radiotracer solutions were administered via the previously installed tail vein catheters and a 2 hour emission image was acquired. Injected radiotracer solutions consisted of either approximately 2001...ri 8F-radiolabeled A02 or 2001...ri 18F-radiolabeled A02 supplemented with 3 mg/kg final concentration of cold, unlabeled A02 Adnectin (based on individual animal weight). All injections were formulated in 200 !IL saline prior to injection. Exact injected doses were calculated by taking direct measurement of the formulated dose and subtracting the radioactivity remaining in the syringe and the tail vein catheter.
[000220] Images were reconstructed using a maximum a posteriori (MAP) algorithm with attenuation correction using the collected transmission images and corrected for radioisotope decay. In the final images, regions of interest (ROIs) were drawn around the tumor boundary using ASIPro software (Siemens Preclinical Solutions). Time-activity curves were calculated for each ROI to yield a quantitative view of radiotracer within the tumor volume over the course of the 2 hour emission image. For final comparison, individual time-activity curves were normalized based on the injected radiotracer dose for each specific animal. Radiotracer uptake was compared across tumors using the final 10 minutes of each time-activity curve (1 hour 50 minutes ¨ 2h post-radiotracer injection). Using this methodology, radiotracer uptake in PD-L1(+) L2987 xenografts was 3.05x that seen PD-L1(-) HT-29 xenografts in animals receiving only the 64Cu-A01 radiotracer. In animals co-injected with the 18F-radiolabeled A02 radiotracer and 3 mg/kg unlabeled A02 Adnectin uptake in the PD-L1(+) xenografts was only 1.04x that seen in PD-L1(-) HT-29 xenografts.
[000221] For some studies, animals were sacrificed via cervical dislocation immediately following imaging. Necropsy was then performed on the animals, and individual tissues were collected (blood, heart, lung, liver, spleen, kidney, muscle, stomach, bone, L2987 tumor, and HT-29 tumor) into pre-weighed tubes. All tissues were then weighed again to determine the weight of each tissue. The radioactivity in each tissue was then directly measured ex vivo using a Perkin-Elmer Wizard3 gamma counter. For all tissues, measured values in counts per minute (CPM) were normalized to the injected radioactive dose for the individual animals and corrected for radioactive decay. These results were then plotted to show the biodistribution of the radiotracer as shown in Figure 2.
[000222] These results demonstrate clear differential uptake of the radiotracer in PD-Li (+) L2987 xenografts compared to PD-L1(-) HT-29 xenografts. Furthermore, the only tissue with higher PD-Li uptake was the kidney, which is expected as clearance of the radiolabeled A02-4PEG-DBCO Adnectin is expected to be via kidney filtration based on the molecular weight of the molecule.
[000223] Taken together, these results provide direct visualization of differentiation of PD-L1(+) versus PD-L1(-) xenograft tumors in vivo. Specificity was further demonstrated by co-injection of 3 mg/kg unlabeled anti-PD-Li A02 adnectin, resulting in a reduction of radiotracer uptake in PD-L1(+) tumors to the level of PD-L1(-) xenografts. A
maximum radiotracer uptake ratio of 3.53:1 in hPD-L1(+) L2987 xenografts vs. hPD-L1(-) xenografts using the 18F-A02 Adnectin radiotracer was obtained. This further validates the use of anti-PD-Li adnectins for visualization of PD-Li tissue expression using PET imaging.
Similar experiments using 18F as the radionuclide were conducted in mice, and similar results were obtained, reaching.

In vivo Imaging in Cynomolgus Monkeys [000224] The 18F-radiolabeled EOlimaging agents also showed similar results when performed in cynomolgus monkeys. In these studies, the 18F-E01 anti-PD-L1, produced as described in Example 9, was tested for the ability to produce high-contrast images in cynomolgus monkeys. The anti-PD-Li adnectins described here maintain high affinity for cynomolgus PD-Li (but have low affinity for rodent PD-L1). Furthermore, as the cynomolgus monkeys do not contain PD-L1(+) tumors as in mouse models, imaging performance was assessed primarily on the background levels measured in the images in the context of endogenous PD-Li expression (with low background enabling the potential for high-sensitivity detection of PD-L1(+) tissues). In these studies, background levels in the resulting PET images were very low, with notable radiotracer accumulation noted mainly in the kidneys, spleen, and bladder.
[000225] Cynomolgus male monkeys with a previously installed vascular access port (VAP) were anesthetized with 0.02 mg/kg atropine, 5 mg/kg Telazol and 0.01 mg/kg buprenorphine I.M. (all drawn into a single syringe). An i.v. catheter is then placed in the cephalic vessel for fluid administration during the imaging procedure to maintain hydration.
Animals were intubated with an endotracheal tube - usually 3.0 mm and transferred to the imaging bed of a microPET F22OTM PET instrument (Siemens Preclinical Solutions, Knoxville, TN).
Anesthesia was maintained with isoflurane and oxygen and I.V. fluids (LRS) were administered at a rate of 6 ml/kg/hr during the imaging procedure. As the axial field of view of the microPET F22OTM instrument is only 7.6 cm, images over 5 distinct bed positions were acquired to create a composite image of the animals from just above the heart through approximately the pelvis.
[000226] For each field of view, a 10 minute transmission image was first acquired using a 57Co point source for the purpose of attenuation correction of the final PET
images. Once transmission images were acquired for all bed positions, approximately 1.5 mCi (approximately 0.015 mg/kg) of the 18F-E01 adnectin radiotracer was administered via the installed VAP. Five minute duration emission scans were then sequentially acquired for each bed position, beginning at position 1 centered approximately at the heart and moving toward the pelvis of the animal. Once images were acquired at each position (1 through 5), the imaging bed was moved back to bed position 1 and the process was repeated.
Using this procedure, a total of 5 distinct images were acquired for each bed position over the duration of the imaging study.
[000227] Individual images were reconstructed using a filtered back projection (FBP) algorithm with attenuation correction using the collected transmission images and corrected for radioisotope decay. Final composite images were then produced by aligning images from all 5 bed positions obtained from a single pass (i.e. a single composite image was produced from each set of sequential images from bed positions 1 through 5) covering the duration of the imaging study (Figure 3). Final images were visually inspected to note areas of visible radiotracer uptake (i.e. spleen, kidney, bladder) and background tissue (muscle). Background accumulation of 18F-E01 adnectin was very low, with little signal visible in background tissues such as muscle. Additionally, uptake was verified in the spleen, which is believed to be PD-L1(+) based on mRNA expression. Thus, studies in cynomolgus monkeys demonstrate the potential for high-sensitivity PD-Li imaging in the context of endogenous PD-Li.
[000228] In aggregate, PET studies in rodent and cynomolgus monkey show that 18F labeled proteins produced according to the methods described herein provide strong and specific probes for in vivo labeling of target positive tissues with the potential for high-sensitivity detection of tissues with low level target expression.

In vitro autoradiography with [18F]-A02 anti-PD-Li adnectin [000229] Human lung tumor tissues were embedded in OCT and chilled in 2-methylbutane for 2-5 minutes until frozen. The samples were stored in -80 C degree freezer until use.
Human xenograft tissues were also included in the assay. Mice bearing bilateral xenografts were produced by introducing 4x106 PD-L1(+) L2987 cells and 1.5x106 PD-L1(-) HT-29 t cells subcutaneously into opposite flanks of nu/nu mice. Once resulting xenograft tumors reached appropriate size (approx. 200-300mm3) mice were anesthetized with 2%
isoflurane and sacrificed via cervical dislocation. Fresh tumor tissues were excised, immersed into OCT
and chilled in 2-methylbutane for 2-5 minutes until frozen. The tissues were then wrapped in foil/ZIPLOC bag and stored at -80 C until use. For all tissues (human lung tumor and xenografts) sections of 5p.m thickness (collected as 2 sections/slide) were cut using a cryostat, thaw-mounted on glass microscope slides, and allowed to air dry for approximately 30 minutes.
[000230] Blocking studies with cold (unlabeled) A02 adnectin at 0.025 nM, 0.25 nM, 2.5 nM
and 25 nM respectively and 25 nM non-PD-Li binding adnectin were conducted using the following conditions. The individual slides, 1 slide per concentration, were placed in plastic slide cassettes and pre-incubated in Dako serum-free protein block solution for 30 minutes.
Slides were then transferred to glass slide incubation chambers for further incubation.
Separately, a stock solution of 0.25 nM 18F-A02 adnectin was produced by diluting 10.6 p1 of the original stock radioligand solution (7064 nM at the time of experiment) with 300 ml of PBS + 0.5% BSA. From this stock solution, 40 ml was added to each incubation chamber.
One of these chambers contained only the radioligand buffer solution, which is referred to as the total binding section. Other incubation chambers received 40 ml of this stock solution along with the relevant concentration of blocking compound (unlabeled A02 adnectin at 0.025 nM, 0.25 nM, 2.5 nM, or 25 nM or unlabeled adnectin at 25 nM). Slides were incubated in the individual buffer solutions for 1 hour at room temperature to reach maximum binding. After incubation, slides from each treatment group were removed from the incubation solutions and placed in an ice-cold wash buffer (PBS + 0.5% BSA) for 3 minutes and rinsed 4 separate times. Slides were then dried under a stream of cold air for approximately 30 minutes. The air-dried slides were exposed by placing the slides onto an imaging plate (BAS-SR 3545S) overnight at room temperature. The imaging plate was scanned using the bioimaging analyzer (Fujifilm Fluorescent Image Analyzer, FLA-9000).
The pixel size of the autoradiogram images was 100 p.m. Image analysis was performed using the Multi-Gauge software. The regions of interest (ROIs) were drawn to surround the entire tumor tissue in all study groups. Autoradiography signal from tissue-associated radioactivity was quantified from these ROIs.
[000231] The apparent displacement of the 18F-A02 adnectin radioligand when compared to the total binding sections was determined for 4 different concentrations (0.025 nM, 0.25 nM, 2.5 nM and 25 nM) of unlabeled A02 adnectin in both human lung tumor sections as well as human xenograft sections. A dose dependent displacement of 18F-A02 was seen in all tissue sections with the addition of unlabeled A02 adnectin. 25 nM non-PD-Li binding adnectin showed minimal blockade in all tissues compared to total binding (Figure 4A).
[000232] Serial 5 i.tm tissue sections from each tissue were subjected to an anti-human-PD-Li immunohistochemical procedure to verify the level of PD-Li antigen expression in the samples (Figure 4B).
[000233] Taken together, these results provide direct visualization of PD-Li in both human lung tumor samples as well as human xenograft tissues. The level of radioligand binding in the individual tissues corresponds with the intensity of PD-Li staining of frozen sections by IHC. In addition, the dose dependent blockade of the receptor with unlabeled anti-PD-Li A02 adnectin (and lack of blockade with unlabeled non-PD-Li binding adnectin), further validates the use of 18F-A02 for visualization of PD-Li tissue expression using PET imaging.

Automated preparation of [18F]-3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-fluoropyridine according to the general procedure for radiosynthesis using commercial GE TRACERlab FX2 N synthesis unit : K.2.2.2 IJ
N's;";--10"`¨'''=¨=` K2CO3 N

120 *C 0 .--0 mins Procedure:
[000234] The automated synthesis of [18F]-3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine was carried out using a non-cassette type GE TRACERlab FX2 N
Synthesis module. The setup of the synthesis unit is summarized in Table 1 and Figure 6.
The aqueous [18F]-Fluoride solution (2.0 ml, 29.6 GBq/ 800 mCi) was delivered to a Sep-Pak light QMA
[The Sep-Pak light QMA cartridge was pre-conditioned sequentially with 5m1 of 0.5 M
potassium bicarbonate, 5 ml of deionized water, and 5 ml of acetonitrile before use.] Upon completion of this transfer, the aqueous [18F] fluoride was released from the QMA Sep-Pak by the addition of the elution mixture (from "V1") into the reactor. The solvent was evaporated under a gentle stream of nitrogen and vacuum. The solution of precursor (from "V3") was added to the dried cryptand residue and this reaction mixture was heated 120 C
for 10 minutes. Then 4 ml of distilled water (from "V4") was added to the crude reaction mixture in the reactor and the mixture is transferred to the 5 ml sample injection loop of the semi-preparative HPLC via a liquid sensor which controls the end of the loading. The mixture was loaded onto the semi-preparative HPLC column (Luna C18(2). 250x 1 Omm, Phenomenex). A mixture of 35% acetonitrile in an aqueous 0.1% trifluoro acetic acid solution was flushed through the column at a rate of 4.6 ml per minute. The product was collected from this HPLC column into the dilution flask which contained 15 ml distilled water and its entire contents were transferred to a tC18 1 gram, solid phase extraction cartridge. 352 mCi (13 GBq) of [18F]-3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine was released from this cartridge ( from "V14") with 3 ml of ethanol and may be used to generate 18F labeled biologic products by taking advantage of "click" azide-alkyne reaction with the appropriate biologic containing an alkynes.
Table 1 Vial 1 (V1) 16 mg K.2.2.2, 3 mg Potassium carbonate, dissolved in 0.1 ml of distilled water and 1.4 ml of acetonitrile Vial 3 (V3) 2 mg 3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-nitropyridine in 0.5 ml DMSO
Vial 4 (V4) 4 ml of distilled water Vial 14 (V14) 3 ml of 100% ethanol Dilution Flask 15 ml of distilled water Cartridge 1 (C1) tC18 6 cc lg sep pack HPLC Column Luna C18(2), 250x1Omm, 5 lam, Phenomenex HPLC Solvent 35% acetonitrile in an aqueous 0.1% trifluoroacetitic acid solution HPLC flow 4.6 ml/min Automated preparation of [18F]-3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-fluoropyridine according to the general procedure for radiosynthesis using a commercial IBA Synthera synthesis unit .t I K.2.2.2 N , r' mins Procedure:
[000235] The automated synthesis of [18F]-3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine was carried out using a cassette type IBA Synthera Synthesis module and an appropriately assembled integrator fluidic processor kit. The integrator fluidic processor (IFP) kit was loaded with appropriate precursors for this synthesis and is summarized in Table 2.The purification was performed on an Varian HPLC unit. The filling of the injection loop of the HPLC was controlled by a steady stream of nitrogen on the HPLC
unit. The setup of both automates are summarized in Table 2. The aqueous [18F]-Fluoride solution (2.0 ml, 29.6 GBq/ 800 mCi) was delivered to a Sep-Pak light QMA [The Sep-Pak light QMA

cartridge was pre-conditioned sequentially with 5m1 of 0.5 M potassium bicarbonate, 5 ml of deionized water, and 5 ml of acetonitrile before use.] Upon completion of this transfer, the aqueous [18F] fluoride was released from the QMA Sep-Pak by the addition of the elution mixture (from "V1") into the reactor. The solvent was evaporated under a gentle stream of nitrogen and vacuum. The solution of precursor (from "V2") was added to the dried cryptand residue and this reaction mixture was heated 120 C for 10 minutes. Then 3 ml of distilled water (from "V4") was added to the crude reaction mixture in the reactor and the mixture was transferred to the 5 ml sample injection loop of the semi-preparative HPLC via a liquid sensor which controls the end of the loading. The mixture was loaded onto the semi-preparative HPLC column (Luna C18(2). 250x 1 Omm, Phenomenex). A mixture of 35%
acetonitrile in an aqueous 0.1% trifluoroacetic acid solution was flushed through the column at a rate of 4.6 ml per minute. The product was collected from this HPLC
column into the dilution flask which contained 15 ml distilled water and its entire contents were transferred to a tC18 1 gram, solid phase extraction cartridge. 325 mCi ( 12 GBq) of [18F]-3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-2-fluoropyridine was released from this cartridge with 3 ml of ethanol and may be used to generate 18F labeled biologic products by taking advantage of "click" azide-alkyne reaction with the appropriate biologic containing an alkynes.
Table 2 Vial 1 (V1) 22 mg K.2.2.2, 4 mg Potassium carbonate, dissolved in 0.3 ml of distilled water and 0.3 ml of acetonitrile Vial 2 (V2) 2 mg 3-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-nitropyridine in 0.5 ml DMSO
Vial 4 (V4) 3 ml of distilled water Dilution Flask 15 ml of distilled water Cartridge 1 (Cl) tC18 6 cc lg sep pack HPLC Column Luna C18(2), 250x1Omm, 5 Om, Phenomenex HPLC Solvent 35% acetonitrile in an aqueous 0.1% trifluoroacetitic acid solution HPLC flow 4.6 ml/min Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims.

Claims (87)

WHAT IS CLAIMED IS:

1. A compound comprising an 18F-radiolabeled prosthetic group having the structure or a pharmaceutically acceptable salt thereof, wherein x is an integer from 1 to 8 and the 18F-radiolabel is attached ortho to the N atom of the pyridine.

2. The compound of claim 1, wherein the 18F-radiolabeled prosthetic group has the structure or a pharmaceutically acceptable salt thereof.

3. The compound of claims 1 or 2, wherein the [O(CH2)2]x moiety is present in the 1-3 configuration relative to the nitrogen on the pyridine ring.

4. The compound of claims 1 or 2, wherein the [O(CH2)2]x moiety is present in the 1-2 configuration relative to the nitrogen on the pyridine ring.

5. The compound of claims 1 or 2, wherein the [O(CH2)2]x moiety is present in the 1-4 configuration relative to the nitrogen on the pyridine ring.

6. The compound of claim 1, wherein the 18F-radiolabeled prosthetic group has the structure or a pharmaceutically acceptable salt thereof.

7. The compound of any one of the preceding claims, wherein x is an integer from 2 to 6, or a pharmaceutically acceptable salt thereof.

8. The compound of claim 4, wherein x is an integer from 3 to 5, or a pharmaceutically acceptable salt thereof.

9. The compound of claim 4, wherein x is 4, or a pharmaceutically acceptable salt thereof.

10. The compound of claim 1, wherein the 18F-radiolabeled prosthetic group has the structure or a pharmaceutically acceptable salt thereof.

11. The compound of any one of the preceding claims, wherein the pyridine ring comprises an additional substituent which does not interfere with the fluorination reaction.

12. The compound of claim 11, wherein the substituent on the pyridine ring is a C1-6 alkyl.

13. The compound of claim 12, wherein the substituent is methyl, ethyl or propyl.

14. A compound comprising an 18F-radiolabeled prosthetic group having the structure wherein "OPEG" is [O(CH2)2]x, and x is an integer from 1 to 8, or a pharmaceutically acceptable salt thereof.

15. The compound of claim 14, wherein x is an integer from 2 to 6, or a pharmaceutically acceptable salt thereof.

16. The compound of claim 14, wherein x is an integer from 3 to 5, or a pharmaceutically acceptable salt thereof.

17. The compound of claim 15, wherein x is 4, or a pharmaceutically acceptable salt thereof.

18. The compound or pharmaceutically acceptable salt thereof of any one of the preceding claims which is water soluble.

19. The compound or pharmaceutically acceptable salt thereof of any one of the preceding claims which is not volatile.

20. An 18F-radiolabeled protein-based probe comprising 18F-radiolabeled prosthetic group, a bifunctional conjugating (BFC) moiety and a protein with the following structure, wherein the 18F is ortho to the N atom, x is an integer from 1 to 8, or pharmaceutically acceptable salt thereof.

21. The 18F-radiolabeled protein-based probe of claim 20, wherein 18F-radiolabeled prosthetic group has the following structure,

22. The 18F-radiolabeled protein-based probe of claims 20 or 21, wherein the [O(CH2)2]x moiety is present in the 1-3 configuration relative to the nitrogen on the pyridine ring.

23. The 18F-radiolabeled protein-based probe of claims 20 or 21, wherein the [O(CH2)2]x moiety is present in the 1-2 configuration relative to the nitrogen on the pyridine ring.

24. The 18F-radiolabeled protein-based probe of claims 20 or 21, wherein the [O(CH2)2]x moiety is present in the 1-4 configuration relative to the nitrogen on the pyridine ring.

25. The 18F-radiolabeled protein-based probe of claim 20, wherein 18F-radiolabeled prosthetic group has the following structure,

26. The 18F-radiolabeled protein-based probe of any one of the claims 20 to 25, wherein x is an integer from 2 to 6.

27. The 18F-radiolabeled protein-based probe of claim 26, wherein x is an integer from 3 to 5.

28. The 18F-radiolabeled protein-based probe of claim 26, wherein x is 4.

29. The 18F-radiolabeled protein-based probe of any one of claims 20-28, wherein the [O(CH2)2]x moiety is present in the 1-3 configuration relative to the nitrogen on the pyridine ring.

30. The 18F-radiolabeled protein-based probe of any one of the claims 20-29, wherein the pyridine ring comprises an additional substituent which does not interfere with the fluorination reaction.

31. The 18F-radiolabeled protein-based probe of claim 30, wherein the substituent on the pyridine ring is a C1-6 alkyl.

32. The 18F-radiolabeled protein-based probe of claim 31, wherein the substituent is methyl, ethyl or propyl,

33. The 18F-radiolabeled protein-based probe of claim 20, wherein the 18F-radiolabeled prosthetic group has the structure

34. An "F-radiolabeled protein-based probe comprising 18F-radiolabeled prosthetic group, a bifunctional conjugating (BFC) moiety and a protein with the following structure wherein "OPEG" is [O(CH2)2]x, and x is an integer from 1 to 8, or a pharmaceutically acceptable salt thereof.

35. The 18F-radiolabeled protein-based probe of claim 34, wherein x is an integer from 2 to 6, or a pharmaceutically acceptable salt thereof.

36. The 18F-radiolabeled protein-based probe of claim 34, wherein x is an integer from 3 to 5, or a pharmaceutically acceptable salt thereof.

37. The 18F-radiolabeled protein-based probe of claim 34, wherein x is 4, or a pharmaceutically acceptable salt thereof.

38. The 18F-radiolabeled protein-based probe of any one of claims 20 to 37, wherein the BFC is a cyclooctyne comprising a reactive group that forms a covalent bond with an amine, carboxyl, carbonyl or thiol functional group on the protein.

39. The 18F-radiolabeled protein-based probe of claim 38, wherein the cyclooctyne is selected from the group consisting of dibenzocyclooctyne (DIBO), biarylazacyclooctynone (BARAC), dimethoxyazacyclooctyne (DIMAC) and dibenzocyclooctyne (DBCO).

40. The 18F-radiolabeled protein-based probe of claim 39, wherein the cyclooctyne is DBCO.

41. The 18F-radiolabeled protein-based probe of any one of claims 20 to 39, wherein the BFC further comprises a polyethylene glycol (PEG)y spacer arm, wherein y is an integer from 1 to 8.

42. The 18F-radiolabeled protein-based probe of 41, wherein y is an integer from 2 to 6.

43. 18F-radiolabeled protein-based probe of 41, wherein y is 4 or 5.

44. The 18F-radiolabeled protein-based probe of claim 39, wherein the BFC
is DBCO-PEG4-NHS-Ester, DBCO-Sulfo-NHS-Ester, DBCO-PEG4-Acid, DBCO-PEG4-Amine or DBCO-PEG4-Maleimide.

45. The 18F-radiolabeled protein-based probe of claim 44, wherein the BFC
is DBCO-PEG4-Maleimide.

46. The 18F-radiolabeled protein-based probe of claim 45, having the following structure, wherein the maleimide group of the BFC is covalently linked to the thiol group on a cysteine residue of the protein.

47. The 18F-radiolabeled protein-based probe of claim 46, wherein the cysteine residue is at the C-terminus of the protein.

48. The 18F-radiolabeled protein-based probe of any one of claims 20 to 47, wherein the protein portion of the probe binds to a biological molecule associated with a disease.

49. The 18F-radiolabeled protein-based probe of claim 48, wherein the disease is selected from the group consisting of solid cancers, hematopoietic cancers, hematological cancers, autoimmune disease, neurodegenerative disease, cardiovascular disease, and pathogenic infection.

50. The 18F-radiolabeled protein-based probe of any one of claims 20 to 49, wherein the protein portion of the probe comprises a ligand.

51. The 18F-radiolabeled protein-based probe of any one of claims 20 to 50, wherein the protein portion of the probe comprises an antibody or antibody fragment.

52. The 18F-radiolabeled protein-based probe of any one of claims 20 to 50, wherein the protein portion of the probe comprises a fibronectin based scaffold (FBS).

53. The 18F-radiolabeled protein-based probe of any one of claims 20 to 52, wherein the probe binds to a tumor-associated antigen.

54. The 18F-radiolabeled protein-based probe of any one of claims 20 to 52, wherein the probe binds to a protein present on a pathogenic organism.

55. The 18F-radiolabeled protein-based probe of claim 54, wherein the pathogenic organism is a virus, bacterium or fungus.

56. A method of obtaining an image of an 18F-radiolabeled protein-based probe of any one of claims 20 to 55, the method comprising, a) administering the 18F-radiolabeled protein-based probe to a subject; and b) imaging in vivo the distribution of the 18F-radiolabeled protein-based probe by positron emission tomography (PET) scanning.

57. The method of claim 56, wherein the imaged distribution of the 18F-radiolabeled protein-based probe is indicative of the presence or absence of a disease.

58. A method of diagnosing the presence of a disease in a subject, the method comprising (a) administering to a subject in need thereof an 18F-radiolabeled protein-based probe of any one of claims 20 to 55 which binds to a target molecule associated with the presence of the disease; and (b) obtaining an radio-image of at least a portion of the subject to detect the presence or absence of the 18F-radiolabeled protein-based probe;
wherein the presence and location of the 18F-radiolabeled protein-based probe above background is indicative of the presence and location of the disease.

59. A method of monitoring the progress of a disease in a subject, the method comprising (a) administering to a subject in need thereof an 18F-radiolabeled protein-based probe of any one of claims 20 to 55 which binds to a target molecule associated with the presence of the disease at a first time point and obtaining an image of at least a portion of the subject to determine the amount of the diseased cells or tissue; and (b) administering to the subject the 18F-radiolabeled protein-based probe at one or more subsequent time points and obtaining an image of at least a portion of the subject at each time point; wherein the dimension and location of the diseased cells or tissue at each time point is indicative of the progress of the disease.

60. A method of quantifying diseased cells or tissues in a subject, the method comprising (a) administering to a subject having diseased cells or tissues an 18F-radiolabeled protein-based probe of any one of claims 20 to 55 which binds to a target molecule located with the diseased cells or tissues; and (b) detecting radioactive emissions of the 18F in the diseased cells or tissue, wherein the level and distribution of the radioactive emissions in the diseased cells or tissues is a quantitative measure of the diseased cells or tissues.

61. The method of any one of claims 58 to 60, wherein the disease is selected from the group consisting of solid cancers, hematopoietic cancers, hematological cancers, autoimmune disease, neurodegenerative disease cardiovascular disease, and pathogenic infection.

62. A method of obtaining a quantitative image of tissues or cells expressing a target protein, the method comprising contacting the cells or tissue with an 18F-radiolabeled protein-based probe of any one of claims 20 to 55 which binds to the target protein, and detecting or quantifying the tissue expressing the target protein using positron emission tomography (PET).

63. A method of screening for an agent for treating a disease comprising the steps of (a) contacting cells expressing a target protein associated with the disease with an 18F-radiolabeled protein-based probe of any one of claims 20 to 55 which binds to the target protein in.the presence and absence of a candidate agent; and (b) imaging the cells in the presence and absence of the candidate agent using positron emission tomography (PET), wherein a decrease in the amount of radioactive emissions in the presence of the candidate agent is indicative of that the agent binds to the target protein.

64. The method any one of claims 56 to 63, wherein the 18F-radiolabeled protein-based probe comprises a ligand.

65. The method any one of claims 56 to 63, wherein the 18F-radiolabeled protein-based probe comprises an antibody or antibody fragment.

66. The method any one of claims 56 to 63, wherein the 18F-radiolabeled protein-based probe comprises a fibronectin based scaffold (FBS).

67. The method any one of claims 56 to 66, wherein the 18F-radiolabeled protein-based probe binds to a tumor-associated antigen.

68. The method any one of claims 56 to 66, wherein 18F-radiolabeled protein-based probe binds to a protein present on a pathogenic organism.

69. The method of claim 68, wherein the pathogenic organism is a virus, bacterium or fungus.

70. A pharmaceutical composition comprising the 18F-radiolabeled protein-based probe of any one of claims 20 to 55.

71. A kit comprising the reaction precursors for use in producing the 18F-radiolabeled protein-based probe of any one of claim 20 to 55, and instructions for producing the 18F-radiolabeled protein-based probe.

72. A method for preparing an 18F-radiolabeled compound with the following structure, wherein x is an integer from 1 to 8, the method comprising the steps of (a) providing a solution of a compound a with the following structure:
a wherein x is an integer from 1 to 8, and R is NO2, Br, F or and is ortho to the N atom of the pyridine ring;
(b) providing a mixture of 18F in 18Owater, 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane and a weak base;
c) drying the mixture from step b) to form a solid; and d) reacting the solution from step a) with the solid from step c) to form the 18F-labeled compound.

73. The method of claim 72, wherein the 18F-radiolabeled compound has the structure

74. The compound of claims 72 or 73, wherein the [O(CH2)2]x moiety is present in the 1-3 configuration relative to the nitrogen on the pyridine ring.

75. The compound of claims 72 or 73, wherein the [O(CH2)2]x moiety is present in the 1-2 configuration relative to the nitrogen on the pyridine ring.

76. The compound of claims 72 or 73, wherein the [O(CH2)2]x moiety is present in the 1-4 configuration relative to the nitrogen on the pyridine ring.

77. The method of claim 73, wherein the 18F-radiolabeled compound has the structure

78. The method of any one of claims 72 to 77, wherein x is an integer from 2 to 6.

79. The method of claim 78, wherein x is an integer from 3 to 5.

80. The method of claim 78, wherein x is 4.

81. The method of claim 77, wherein the 18F-radiolabeled prosthetic group has the structure

82. A method for preparing an 18F-labeled compound of the structure which comprises a) providing a solution of the compound of the structure where X is NO2, Br or b) providing a mixture of 18F in Owater, 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane and weak base;
c) drying the mixture from step b) to form a solid; and d) reacting the solution from step a) with the solid from step c) to form the 18F-labeled compound.

83. The method of claim 72, wherein R is NO2 in the starting compound from step a)

84. A method of producing the 18F-radiolabeled protein-based probe of any one of claims 20 to 55, the method comprising coupling the 18F-radiolabeled prosthetic group of any one of claims 1 to 19 with a protein comprising a bifunctional conjugating (BFC) moiety using a bioorthogonal click reaction.

85. The method of claim 84, wherein the bioorthogonal click reaction is metal free.

86. The method of claim 84 or 85, wherein the BFC coupled to the protein comprises a cyclooctyne.

87. The method of claim 86, wherein the cyclooctyne is DBCO-PEG4-Maleimide.